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The medicinal chemistry of the isomers of the cyclic dipeptide: cyclo(Trp-Pro)
- Authors: Jamie, Hajierah
- Date: 2002
- Subjects: Pharmaceutical chemistry , Cyclic compounds
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:11023 , http://hdl.handle.net/10948/281 , Pharmaceutical chemistry , Cyclic compounds
- Description: The isomers of cyclo(Trp-Pro) (cyclo(L-Trp-L-Pro), cyclo(L-Trp-D-Pro), cyclo(D-Trp-LPro) and cyclo(D-Trp-D-Pro)) have been successfully synthesized and screened for biological activity. High percentage yields were obtained by using the three phase synthesis system, which involves the synthesis of the intermediate protected linear dipeptides, followed by the removal of the protecting Boc groups. This step is followed by cyclization and crystallization of the isomers. The diketopiperazines rings of cyclo(L-Trp-L-Pro) and cyclo(D-Trp-D-Pro) contain cisamide bonds, while cyclo(L-Trp-D-Pro) and cyclo(D-Trp-L-Pro) contain trans-amide bonds. These bonds govern the conformation of the diketopiperazines ring. The isomers have shown different degrees of biological activity, possibly as a result of the orientation of the side chain of tryptophan and this difference in conformation, leading to varying interactions between isomer and a range of receptors. Under experimental conditions, 10-3 M cyclo(L-Trp-D-Pro) and cyclo(D-Trp-L-Pro) showed effective anticancer activity against the cervical cancer cell line, HeLa, resulting in a <50% reduction in cell viability. Cytotoxicity screening with cyclo(D-Trp-L-Pro) indicated that it was hepatocyte-specific in its toxicity, whilst the other isomers were cytotoxic against the other cell types tested. At 1mg/ml, cyclo(L-Trp-L-Pro) proved to be an effective antimicrobial agent against Gram positive bacteria, while cyclo(L-Trp-DPro) effectively inhibited the growth of the Gram negative bacteria, Esherichia coli. Cyclo(D-Trp-L-Pro) proved to be effective against Streptococcus, while cyclo(D-Trp-DPro) effectively reduced viability of the yeast, Candida albicans. Cyclo(D-Trp-L-Pro) was the only isomer to show Ca2+-channel antagonism, whilst the other isomers resulted in opening of the Ca2+-channel. No effects were observed on K+-channel activity for all the isomers tested. The isomers also proved to be valuable antiarrhythmic agents by effectively reducing the time spent in ventricular tachycardia and arrhythmia, as well as decreasing the time for the heart rate to return to a normal sinus rhythm. Furthermore, cyclo(L-Trp-D-Pro) showed positive chronotropic activity, while cyclo(D-Trp-L-Pro) ii showed negative chronotropic activity. In addition, cyclo(L-Trp-D-Pro) and cyclo(D-Trp- L-Pro) also increased the coronary flow rate. 0.125 1 mM Cyclo(L-Trp-D-Pro) decreased aggregation in washed platelets induced by thrombin. All isomers increased adhesion to an artificial surface when the platelets were stimulated by ADP, yet caused reduced adhesion when the platelets were stimulated by thrombin. These results prove the potential of these compounds as novel agents in a range of biological fields, indicating that a combination of L- and D- amino acids may prove more effective than an agent consisting solely of L-amino acids.
- Full Text:
- Authors: Jamie, Hajierah
- Date: 2002
- Subjects: Pharmaceutical chemistry , Cyclic compounds
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:11023 , http://hdl.handle.net/10948/281 , Pharmaceutical chemistry , Cyclic compounds
- Description: The isomers of cyclo(Trp-Pro) (cyclo(L-Trp-L-Pro), cyclo(L-Trp-D-Pro), cyclo(D-Trp-LPro) and cyclo(D-Trp-D-Pro)) have been successfully synthesized and screened for biological activity. High percentage yields were obtained by using the three phase synthesis system, which involves the synthesis of the intermediate protected linear dipeptides, followed by the removal of the protecting Boc groups. This step is followed by cyclization and crystallization of the isomers. The diketopiperazines rings of cyclo(L-Trp-L-Pro) and cyclo(D-Trp-D-Pro) contain cisamide bonds, while cyclo(L-Trp-D-Pro) and cyclo(D-Trp-L-Pro) contain trans-amide bonds. These bonds govern the conformation of the diketopiperazines ring. The isomers have shown different degrees of biological activity, possibly as a result of the orientation of the side chain of tryptophan and this difference in conformation, leading to varying interactions between isomer and a range of receptors. Under experimental conditions, 10-3 M cyclo(L-Trp-D-Pro) and cyclo(D-Trp-L-Pro) showed effective anticancer activity against the cervical cancer cell line, HeLa, resulting in a <50% reduction in cell viability. Cytotoxicity screening with cyclo(D-Trp-L-Pro) indicated that it was hepatocyte-specific in its toxicity, whilst the other isomers were cytotoxic against the other cell types tested. At 1mg/ml, cyclo(L-Trp-L-Pro) proved to be an effective antimicrobial agent against Gram positive bacteria, while cyclo(L-Trp-DPro) effectively inhibited the growth of the Gram negative bacteria, Esherichia coli. Cyclo(D-Trp-L-Pro) proved to be effective against Streptococcus, while cyclo(D-Trp-DPro) effectively reduced viability of the yeast, Candida albicans. Cyclo(D-Trp-L-Pro) was the only isomer to show Ca2+-channel antagonism, whilst the other isomers resulted in opening of the Ca2+-channel. No effects were observed on K+-channel activity for all the isomers tested. The isomers also proved to be valuable antiarrhythmic agents by effectively reducing the time spent in ventricular tachycardia and arrhythmia, as well as decreasing the time for the heart rate to return to a normal sinus rhythm. Furthermore, cyclo(L-Trp-D-Pro) showed positive chronotropic activity, while cyclo(D-Trp-L-Pro) ii showed negative chronotropic activity. In addition, cyclo(L-Trp-D-Pro) and cyclo(D-Trp- L-Pro) also increased the coronary flow rate. 0.125 1 mM Cyclo(L-Trp-D-Pro) decreased aggregation in washed platelets induced by thrombin. All isomers increased adhesion to an artificial surface when the platelets were stimulated by ADP, yet caused reduced adhesion when the platelets were stimulated by thrombin. These results prove the potential of these compounds as novel agents in a range of biological fields, indicating that a combination of L- and D- amino acids may prove more effective than an agent consisting solely of L-amino acids.
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An investigation into the introduction of process analytical technology, using near infrared analysis, to selected pharmaceutical processes
- Authors: Naicker, Krishnaveni
- Date: 2007
- Subjects: Near infrared spectroscopy , Pharmaceutical chemistry
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10153 , http://hdl.handle.net/10948/577 , http://hdl.handle.net/10948/d1011710 , Near infrared spectroscopy , Pharmaceutical chemistry
- Description: Introduction: Process analytical technologies are systems for the analysis and control of manufacturing processes to assure acceptable end-product quality. This is achieved by timely measurements of critical parameters and performance attributes of raw material and in-process material and processes. The introduction of process analytical technology using near infrared analysis was investigated in three areas, namely incoming raw material analysis, blend uniformity analysis and moisture determination in the fluid bed dryer. Methodology: Incoming raw material identification - The FOSS XDS rapid content analyzer was used for the development of a NIR method for the identification and material qualification of starch maize and lactose monohydrate. Blend uniformity analysis – The SP15 Laboratory Blender fitted with near infrared probe was utilized for the study. Two types of blend experiments were designed to monitor the distribution of magnesium stearate (lubricant) in the blend, namely, a powder blend utilizing lactose monohydrate and a granule blend utilizing Ridaq® granule. Software methods were developed to monitor the standard deviation of the absorbance at the wavelengths that were specific for lactose monohydrate, Ridaq® granule and magnesium stearate. To confirm the prediction of end-point using near infrared, results were verified using an atomic absorption method for magnesium stearate. The blends were sampled at the selected time intervals corresponding to three states of the blend, namely, before end-point, at end-point and after end-point using a sampling plan. An additional six blends were conducted for the granule blend and sampled when the standard deviation had reached a value below 3 x 10-6 at the magnesium stearate wavelength at four consecutive data points (standard deviation value extrapolated from blends carried out to predetermined time intervals). Moisture determination in the fluid bed dryer – Moisture values for two products (Product A and Product B) were retrospectively collected from past production batches. A process capability study was conducted on the moisture values to determine if the current process was in a state of control. Results and Discussion: Incoming raw material identification – The algorithms used for the spectral library were able to distinguish between the raw materials selected. The spectral library positively identified the starch maize and lactose monohydrate samples that were not present in the library. The negative challenge with pregelatinised starch and tablettose demonstrated that the spectral library was able to differentiate between closely related compounds. Blend uniformity analysis – Blends sampled at the predetermined time intervals demonstrated a homogeneous state when the standard deviation of the absorbance was low and a non-homogeneous state when the standard deviation of the absorbance was high, thus near infrared prediction on the state of the blend was confirmed by the standard analytical methods. The series of Ridaq® granule and magnesium stearate blends sampled when the standard deviation was below 3 x 10-6 were homogeneous with the exception of one blend that was marginally out of specification. Blend durations were significantly lower than the standard blend durations used in the facility and ranged from 112 to 198 seconds. Moisture determination in the fluid bed dryer – From the process capability study of the two products it was noted that Product A is stable but can still be optimized while Product B is at a desirable state. The statistical evaluation of the moisture values for Product A and Product B demonstrated that the use of the product temperature to monitor the moisture gave consistent results. The current process is stable and capable of producing repeatable results although near infrared provides a means for continuously monitoring the product moisture and allows one to take action to prevent over-drying or under-drying. Conclusion: From the investigations conducted, it can be seen that there is definitely a niche for process analytical technology at this pharmaceutical company. The implementation is a gradual process of change, which may take time, probably several years (Heinze & Hansen 2005).
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- Authors: Naicker, Krishnaveni
- Date: 2007
- Subjects: Near infrared spectroscopy , Pharmaceutical chemistry
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10153 , http://hdl.handle.net/10948/577 , http://hdl.handle.net/10948/d1011710 , Near infrared spectroscopy , Pharmaceutical chemistry
- Description: Introduction: Process analytical technologies are systems for the analysis and control of manufacturing processes to assure acceptable end-product quality. This is achieved by timely measurements of critical parameters and performance attributes of raw material and in-process material and processes. The introduction of process analytical technology using near infrared analysis was investigated in three areas, namely incoming raw material analysis, blend uniformity analysis and moisture determination in the fluid bed dryer. Methodology: Incoming raw material identification - The FOSS XDS rapid content analyzer was used for the development of a NIR method for the identification and material qualification of starch maize and lactose monohydrate. Blend uniformity analysis – The SP15 Laboratory Blender fitted with near infrared probe was utilized for the study. Two types of blend experiments were designed to monitor the distribution of magnesium stearate (lubricant) in the blend, namely, a powder blend utilizing lactose monohydrate and a granule blend utilizing Ridaq® granule. Software methods were developed to monitor the standard deviation of the absorbance at the wavelengths that were specific for lactose monohydrate, Ridaq® granule and magnesium stearate. To confirm the prediction of end-point using near infrared, results were verified using an atomic absorption method for magnesium stearate. The blends were sampled at the selected time intervals corresponding to three states of the blend, namely, before end-point, at end-point and after end-point using a sampling plan. An additional six blends were conducted for the granule blend and sampled when the standard deviation had reached a value below 3 x 10-6 at the magnesium stearate wavelength at four consecutive data points (standard deviation value extrapolated from blends carried out to predetermined time intervals). Moisture determination in the fluid bed dryer – Moisture values for two products (Product A and Product B) were retrospectively collected from past production batches. A process capability study was conducted on the moisture values to determine if the current process was in a state of control. Results and Discussion: Incoming raw material identification – The algorithms used for the spectral library were able to distinguish between the raw materials selected. The spectral library positively identified the starch maize and lactose monohydrate samples that were not present in the library. The negative challenge with pregelatinised starch and tablettose demonstrated that the spectral library was able to differentiate between closely related compounds. Blend uniformity analysis – Blends sampled at the predetermined time intervals demonstrated a homogeneous state when the standard deviation of the absorbance was low and a non-homogeneous state when the standard deviation of the absorbance was high, thus near infrared prediction on the state of the blend was confirmed by the standard analytical methods. The series of Ridaq® granule and magnesium stearate blends sampled when the standard deviation was below 3 x 10-6 were homogeneous with the exception of one blend that was marginally out of specification. Blend durations were significantly lower than the standard blend durations used in the facility and ranged from 112 to 198 seconds. Moisture determination in the fluid bed dryer – From the process capability study of the two products it was noted that Product A is stable but can still be optimized while Product B is at a desirable state. The statistical evaluation of the moisture values for Product A and Product B demonstrated that the use of the product temperature to monitor the moisture gave consistent results. The current process is stable and capable of producing repeatable results although near infrared provides a means for continuously monitoring the product moisture and allows one to take action to prevent over-drying or under-drying. Conclusion: From the investigations conducted, it can be seen that there is definitely a niche for process analytical technology at this pharmaceutical company. The implementation is a gradual process of change, which may take time, probably several years (Heinze & Hansen 2005).
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The medicinal chemistry of cyclo (Ser-Ser) and cyclo (Ser-Tyr)
- Authors: Kritzinger, André Louis
- Date: 2007
- Subjects: Cyclic peptides , Peptide drugs -- Therapeutic use , Haematostasis , Pharmaceutical chemistry
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10155 , http://hdl.handle.net/10948/537 , http://hdl.handle.net/10948/d1011712 , Cyclic peptides , Peptide drugs -- Therapeutic use , Haematostasis , Pharmaceutical chemistry
- Description: Cyclic dipeptides are widely used as models for larger peptides because of their simplicity and limited conformational freedom. Some cyclic dipeptides have been shown to produce antiviral, antibiotic and anti-tumour activity (Milne et al., 1998). In this study the cyclic dipeptides, cyclo(Ser-Ser) and cyclo(Ser-Tyr), were synthesised from their corresponding linear precursors using a modified phenolinduced cyclisation procedure. The phenol-induced cyclisation procedure resulted in good yields and purity of the cyclic dipeptides. Quantitative analysis and evaluation of the physicochemical properties of the cyclic dipeptides was achieved by using high-performance liquid chromatography, scanning electron microscopy, thermal analysis and X-ray powder diffraction. The structures of the synthesised cyclic dipeptides were elucidated using infrared spectroscopy, mass spectrometry, nuclear magnetic resonance spectroscopy and molecular modelling. The study aimed to determine the biological activity of cyclo(Ser-Ser) and cyclo(Ser-Tyr) with respect to their anticancer, antimicrobial, haematological and cardiac effects. Anticancer studies revealed that cyclo(Ser-Ser) and cyclo(Ser- Tyr) inhibited the growth of HeLa (cervical cancer), HT-29 (colon cancer) and MCF (breast cancer) cancer cell lines. Both cyclic dipeptides also inhibited the growth of certain selected Gram-positive, Gram-negative and fungal microorganisms in the antimicrobial study. Although the inhibition of growth in the anticancer and antimicrobial studies was statistically significant, the clinical relevance is questionable, since the inhibition produced by both cyclic dipeptides was very limited compared to other pre-existing anticancer and antimicrobial agents. Cyclo(Ser-Tyr) exhibited significant activity in the haematological studies, where it increased the rate of calcium induced-coagulation, and decreased the rate of streptokinase-induced fibrinolysis. Both cyclic dipeptides, however, failed to produce any significant effects on thrombin-substrate binding and ADPinduced platelet aggregation. Cardiac studies revealed that cyclo(Ser-Ser) and especially cyclo(Ser-Tyr) reduced the heart rate, coronary flow rate and ventricular pressure of isolated rat hearts.
- Full Text:
- Authors: Kritzinger, André Louis
- Date: 2007
- Subjects: Cyclic peptides , Peptide drugs -- Therapeutic use , Haematostasis , Pharmaceutical chemistry
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10155 , http://hdl.handle.net/10948/537 , http://hdl.handle.net/10948/d1011712 , Cyclic peptides , Peptide drugs -- Therapeutic use , Haematostasis , Pharmaceutical chemistry
- Description: Cyclic dipeptides are widely used as models for larger peptides because of their simplicity and limited conformational freedom. Some cyclic dipeptides have been shown to produce antiviral, antibiotic and anti-tumour activity (Milne et al., 1998). In this study the cyclic dipeptides, cyclo(Ser-Ser) and cyclo(Ser-Tyr), were synthesised from their corresponding linear precursors using a modified phenolinduced cyclisation procedure. The phenol-induced cyclisation procedure resulted in good yields and purity of the cyclic dipeptides. Quantitative analysis and evaluation of the physicochemical properties of the cyclic dipeptides was achieved by using high-performance liquid chromatography, scanning electron microscopy, thermal analysis and X-ray powder diffraction. The structures of the synthesised cyclic dipeptides were elucidated using infrared spectroscopy, mass spectrometry, nuclear magnetic resonance spectroscopy and molecular modelling. The study aimed to determine the biological activity of cyclo(Ser-Ser) and cyclo(Ser-Tyr) with respect to their anticancer, antimicrobial, haematological and cardiac effects. Anticancer studies revealed that cyclo(Ser-Ser) and cyclo(Ser- Tyr) inhibited the growth of HeLa (cervical cancer), HT-29 (colon cancer) and MCF (breast cancer) cancer cell lines. Both cyclic dipeptides also inhibited the growth of certain selected Gram-positive, Gram-negative and fungal microorganisms in the antimicrobial study. Although the inhibition of growth in the anticancer and antimicrobial studies was statistically significant, the clinical relevance is questionable, since the inhibition produced by both cyclic dipeptides was very limited compared to other pre-existing anticancer and antimicrobial agents. Cyclo(Ser-Tyr) exhibited significant activity in the haematological studies, where it increased the rate of calcium induced-coagulation, and decreased the rate of streptokinase-induced fibrinolysis. Both cyclic dipeptides, however, failed to produce any significant effects on thrombin-substrate binding and ADPinduced platelet aggregation. Cardiac studies revealed that cyclo(Ser-Ser) and especially cyclo(Ser-Tyr) reduced the heart rate, coronary flow rate and ventricular pressure of isolated rat hearts.
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Solid-phase extraction based sample preparation for the determination of drug and organic pollutant residue
- Authors: Pule, Bellah Oreeditse
- Date: 2011 , 2011-02-08
- Subjects: Food contamination , Drugs -- Analysis , Pharmaceutical chemistry , Extraction (Chemistry) , Sorbents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4406 , http://hdl.handle.net/10962/d1006711 , Food contamination , Drugs -- Analysis , Pharmaceutical chemistry , Extraction (Chemistry) , Sorbents
- Description: This thesis presents solid phase extraction (SPE) methodologies based on mixed-mode polymeric sorbents; a mixed mode strong anion exchanger (Agilent SampliQ SAX) and a mixed mode strong cation exchanger (Agilent SampliQ SCX). Furthermore, dispersive-SPE based on a quick, easy, cheap, effective, rugged and safe (QuEChERS) method was assessed for applicability in the determination of drug residues. The mixed-mode polymeric sorbents were evaluated for the simultaneous fractionation of drugs that exhibit diverse polarities with acidic, basic and neutral functionalities in biological matrices (plasma and urine). The polymeric skeleton of these sorbents entails an exchanger group and therefore provides two retention mechanisms, strong cation or anion exchange retention mechanisms with hydrophobic interactions. It was demonstrated that with a sequential elution protocol for sample clean-up analytes were fractionated into acidic, basic and neutral classes. The SAX was employed for analysis of ketoprofen, naproxen (acidic drugs), nortriptyline (basic) and secobarbital (neutral) from urine sample. The SCX was used for fractionating phenobarbital, p-toluamide (acidic), amphetamine, m-toluidine (basic) and acetaminophen (neutral drug) from plasma sample. QuEChERS method was employed for quantitative determination of 16 polycyclic aromatic hydrocarbons (PAHs) from fish fillets and soil; 9 sulfonamides (SAs) from chicken muscles and acrylamide (AA) in cooking oil. The analyte recoveries ranged from 79.6 - 109% with RSDs ranging from 0.06 - 1.9% at three different fortification levels. Good linearity (r2 > 0.9990) was attained for most analytes. The limits of detection and quantification ranged from 0.03 - 0.84 μg/ml and 0.81 - 1.89 μg/ml respectively for analytes in biological samples. LODs and LOQs for analytes in food and environmental samples ranged from 0.02 to 0.39 and 0.25 to 1.30 ng/g respectively.
- Full Text:
- Authors: Pule, Bellah Oreeditse
- Date: 2011 , 2011-02-08
- Subjects: Food contamination , Drugs -- Analysis , Pharmaceutical chemistry , Extraction (Chemistry) , Sorbents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4406 , http://hdl.handle.net/10962/d1006711 , Food contamination , Drugs -- Analysis , Pharmaceutical chemistry , Extraction (Chemistry) , Sorbents
- Description: This thesis presents solid phase extraction (SPE) methodologies based on mixed-mode polymeric sorbents; a mixed mode strong anion exchanger (Agilent SampliQ SAX) and a mixed mode strong cation exchanger (Agilent SampliQ SCX). Furthermore, dispersive-SPE based on a quick, easy, cheap, effective, rugged and safe (QuEChERS) method was assessed for applicability in the determination of drug residues. The mixed-mode polymeric sorbents were evaluated for the simultaneous fractionation of drugs that exhibit diverse polarities with acidic, basic and neutral functionalities in biological matrices (plasma and urine). The polymeric skeleton of these sorbents entails an exchanger group and therefore provides two retention mechanisms, strong cation or anion exchange retention mechanisms with hydrophobic interactions. It was demonstrated that with a sequential elution protocol for sample clean-up analytes were fractionated into acidic, basic and neutral classes. The SAX was employed for analysis of ketoprofen, naproxen (acidic drugs), nortriptyline (basic) and secobarbital (neutral) from urine sample. The SCX was used for fractionating phenobarbital, p-toluamide (acidic), amphetamine, m-toluidine (basic) and acetaminophen (neutral drug) from plasma sample. QuEChERS method was employed for quantitative determination of 16 polycyclic aromatic hydrocarbons (PAHs) from fish fillets and soil; 9 sulfonamides (SAs) from chicken muscles and acrylamide (AA) in cooking oil. The analyte recoveries ranged from 79.6 - 109% with RSDs ranging from 0.06 - 1.9% at three different fortification levels. Good linearity (r2 > 0.9990) was attained for most analytes. The limits of detection and quantification ranged from 0.03 - 0.84 μg/ml and 0.81 - 1.89 μg/ml respectively for analytes in biological samples. LODs and LOQs for analytes in food and environmental samples ranged from 0.02 to 0.39 and 0.25 to 1.30 ng/g respectively.
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The medicinal chemistry of cyclo(D-Phe-2Cl-Pro) and cyclo(Phe-4F-Pro)
- Authors: Ndung'u, Susan Wanjiru
- Date: 2011
- Subjects: Peptide drugs , Cyclic peptides , Pharmaceutical chemistry , Peptides -- Synthesis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/7083 , vital:21223
- Description: Although peptides and proteins are considered as lead compounds for the discovery and development of new therapeutic agents, poor metabolic and physical properties have limited their optimisation as drug candidates (Adessi & Soto, 2002). Research by medicinal chemists however, generated the discovery of structural similarities between some peptides and diketopiperazines and the common occurrence of such compounds in natural products. This discovery initiated the synthesis of diketopiperazines from amino acids in an attempt to bypass the previously mentioned limitations of using peptides as drug candidates (Dinsmore & Beshore, 2002). Diketopiperazines (DKPs) are the simplest form of cyclic dipeptides, and a class of unexplored bioactive peptides that have great potential for the future. The compounds are relatively simple to synthesise and are prevalent in nature (Prasad, 1995). The DKP backbone is rigid and therefore poses conformational constraint on the compounds. This rigidity allows for simple conformational analysis of the compounds and also gives insight into the conformational requirements for interaction with the targets involved in their biological activity. The reduced conformational freedom also increases the receptor specificity and thus the compounds are proposed to have less unfavourable effects (Anteunis, 1978). The aim of the study was to synthesise compounds that would exhibit metabolic stability, receptor specificity and enhanced lipophilicity which would increase the bioavailability of the compounds. This was to be achieved by the introduction of fluorine and chlorine elements into the DKPs. The structure of the DKPs would be altered which in turn would improve the physicochemical properties and the biological activity of the compounds (Naumann, 1999). Cyclo(D-Phe-2Cl-Pro) and cyclo(Phe-4F-Pro) were synthesised using the method of Milne et al. (1992) and by boiling the linear counterparts under reflux in sec-butanol-toluene. The structures of the synthesised DKPs were elucidated using mass spectrometry, nuclear magnetic resonance spectroscopy, infrared spectroscopy and molecular modeling. Qualitative analysis and evaluation of the physicochemical properties of the DKPs were performed using high-performance liquid chromatography, scanning electron microscopy, thermogravimetric analysis, differential scanning calorimetry and x-ray powder diffraction. The study aimed to determine the biological activity of cyclo(D-Phe-2Cl-Pro) and cyclo(Phe-4F-Pro) with respect to their anticancer, antimicrobial, haematological and antidiabetic effects. The anticancer results obtained indicated that the percentage inhibition produced by both DKPs were lower than those proposed by Graz et al. (2000) for proline-containing DKPs where, a greater than 50% inhibition was observed for cyclo(Phe-Pro). Antimicrobial studies revealed that both DKPs demonstrated marginal effects on Gram-positive and Gram-negative organisms but showed significant effects against C. albicans. The haematological studies revealed that cyclo(D-Phe-2Cl-Pro) at a screening concentration of 12.5 mM, significantly decreased the levels of D-dimer (P < 0.0001). The antidiabetics studies showed limited activity of the DKPs in inhibiting the activity of α-glucosidase and α-amylase enzymes.
- Full Text:
- Authors: Ndung'u, Susan Wanjiru
- Date: 2011
- Subjects: Peptide drugs , Cyclic peptides , Pharmaceutical chemistry , Peptides -- Synthesis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/7083 , vital:21223
- Description: Although peptides and proteins are considered as lead compounds for the discovery and development of new therapeutic agents, poor metabolic and physical properties have limited their optimisation as drug candidates (Adessi & Soto, 2002). Research by medicinal chemists however, generated the discovery of structural similarities between some peptides and diketopiperazines and the common occurrence of such compounds in natural products. This discovery initiated the synthesis of diketopiperazines from amino acids in an attempt to bypass the previously mentioned limitations of using peptides as drug candidates (Dinsmore & Beshore, 2002). Diketopiperazines (DKPs) are the simplest form of cyclic dipeptides, and a class of unexplored bioactive peptides that have great potential for the future. The compounds are relatively simple to synthesise and are prevalent in nature (Prasad, 1995). The DKP backbone is rigid and therefore poses conformational constraint on the compounds. This rigidity allows for simple conformational analysis of the compounds and also gives insight into the conformational requirements for interaction with the targets involved in their biological activity. The reduced conformational freedom also increases the receptor specificity and thus the compounds are proposed to have less unfavourable effects (Anteunis, 1978). The aim of the study was to synthesise compounds that would exhibit metabolic stability, receptor specificity and enhanced lipophilicity which would increase the bioavailability of the compounds. This was to be achieved by the introduction of fluorine and chlorine elements into the DKPs. The structure of the DKPs would be altered which in turn would improve the physicochemical properties and the biological activity of the compounds (Naumann, 1999). Cyclo(D-Phe-2Cl-Pro) and cyclo(Phe-4F-Pro) were synthesised using the method of Milne et al. (1992) and by boiling the linear counterparts under reflux in sec-butanol-toluene. The structures of the synthesised DKPs were elucidated using mass spectrometry, nuclear magnetic resonance spectroscopy, infrared spectroscopy and molecular modeling. Qualitative analysis and evaluation of the physicochemical properties of the DKPs were performed using high-performance liquid chromatography, scanning electron microscopy, thermogravimetric analysis, differential scanning calorimetry and x-ray powder diffraction. The study aimed to determine the biological activity of cyclo(D-Phe-2Cl-Pro) and cyclo(Phe-4F-Pro) with respect to their anticancer, antimicrobial, haematological and antidiabetic effects. The anticancer results obtained indicated that the percentage inhibition produced by both DKPs were lower than those proposed by Graz et al. (2000) for proline-containing DKPs where, a greater than 50% inhibition was observed for cyclo(Phe-Pro). Antimicrobial studies revealed that both DKPs demonstrated marginal effects on Gram-positive and Gram-negative organisms but showed significant effects against C. albicans. The haematological studies revealed that cyclo(D-Phe-2Cl-Pro) at a screening concentration of 12.5 mM, significantly decreased the levels of D-dimer (P < 0.0001). The antidiabetics studies showed limited activity of the DKPs in inhibiting the activity of α-glucosidase and α-amylase enzymes.
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Isolation, structural characterisation and evaluation of cytotoxic activity of natural products from selected South African marine red algae
- Authors: Knott, Michael George
- Date: 2012
- Subjects: Marine algae -- South Africa , Red algae -- South Africa , Pharmaceutical chemistry
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3862 , http://hdl.handle.net/10962/d1015460
- Description: The medicinal chemistry of selected marine algae indigenous to South Africa was investigated. Following the isolation and characterisation of a number of new and known compounds, the associated in vitro cytotoxic profiles of these new compounds was investigated. Plocamium maxillosum yielded two new cyclic polyhalogenated monoterpenes which were characterised as 2E-chloromethine-4E-chlorovinyl-4-methyl-5-cyclohexen-1-one (2.1) and 2Z-chloromethine-4E-chlorovinyl-4-methyl-5-cyclohexen-1-one (2.2) on the basis of one and two dimensional NMR spectroscopic data and MS analysis. These compounds were also found to have good cytotoxic activity against breast cancer cell lines. Although these compounds are based on a regular monoterpene skeleton, they represent an uncommon feature not often seen in cyclic halogenated monoterpenes from marine algae. Plocamium robertiae yielded one new cyclic polyhalogenated monoterpene identified as 4,5- dibromo-5-chloromethyl-1-chlorovinyl-2-chloro-methylcyclohexane (2.6) and one known compound called 2,4-dichloro-1-chlorovinyl-1-methylcyclohexane-5-ene or Plocamene D (2.9). Portieria hornemannii was collected from Port Edward in Natal and yielded three new compounds, namely; 3Z-1,6-dibromo-3-(bromomethylidene)-2,7-dichloro-7-methyloctane (3.1), 1E,3Z-1,6-dibromo-3-(bromomethylidene)-7-chloro-7-methyloct-1-ene (3.2), 1Z,3Z- 1,6-dibromo-3-(bromomethylidene)-7-chloro-7-methyloct-1-ene (3.3), and one known compound, namely; 3S,6R-6-bromo-3-(bromomethyl)-3,7-dichloro-7-methyloct-1-ene (3.4). Compounds 3.1 and 3.2 showed no cytotoxic activity against breast cancer cells. Another Portieria hornemannii sample was collected from Noordhoek in the Eastern Cape, it yielded one known compound referred to as 3Z-6-bromo-3-(bromomethylidene)-2,7- dichloro-7-methyloct-1-ene (3.5), as well as one new compound called portieric acid A (3.6) or 5-bromo-2-(bromomethylidene)-6-chloro-6-methylheptanoic acid. Portieric acid A showed slight cytotoxic activity and also represents a new class of compound within the genus Portieria. The isolation of secondary metabolites from the South African red alga, Laurencia glomerata, yielded two known compounds; 7-hydroxylaurene (4.9) and cis-neolaurencenyne (4.12), as well as one chamigrane related compound (4.11). Laurencia flexuosa yielded one known compound called 3Z-bromofucin (4.13). Using 1H NMR, GC and molecular systematics, a novel method for identifying different species of Laurencia was also investigated.
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- Authors: Knott, Michael George
- Date: 2012
- Subjects: Marine algae -- South Africa , Red algae -- South Africa , Pharmaceutical chemistry
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3862 , http://hdl.handle.net/10962/d1015460
- Description: The medicinal chemistry of selected marine algae indigenous to South Africa was investigated. Following the isolation and characterisation of a number of new and known compounds, the associated in vitro cytotoxic profiles of these new compounds was investigated. Plocamium maxillosum yielded two new cyclic polyhalogenated monoterpenes which were characterised as 2E-chloromethine-4E-chlorovinyl-4-methyl-5-cyclohexen-1-one (2.1) and 2Z-chloromethine-4E-chlorovinyl-4-methyl-5-cyclohexen-1-one (2.2) on the basis of one and two dimensional NMR spectroscopic data and MS analysis. These compounds were also found to have good cytotoxic activity against breast cancer cell lines. Although these compounds are based on a regular monoterpene skeleton, they represent an uncommon feature not often seen in cyclic halogenated monoterpenes from marine algae. Plocamium robertiae yielded one new cyclic polyhalogenated monoterpene identified as 4,5- dibromo-5-chloromethyl-1-chlorovinyl-2-chloro-methylcyclohexane (2.6) and one known compound called 2,4-dichloro-1-chlorovinyl-1-methylcyclohexane-5-ene or Plocamene D (2.9). Portieria hornemannii was collected from Port Edward in Natal and yielded three new compounds, namely; 3Z-1,6-dibromo-3-(bromomethylidene)-2,7-dichloro-7-methyloctane (3.1), 1E,3Z-1,6-dibromo-3-(bromomethylidene)-7-chloro-7-methyloct-1-ene (3.2), 1Z,3Z- 1,6-dibromo-3-(bromomethylidene)-7-chloro-7-methyloct-1-ene (3.3), and one known compound, namely; 3S,6R-6-bromo-3-(bromomethyl)-3,7-dichloro-7-methyloct-1-ene (3.4). Compounds 3.1 and 3.2 showed no cytotoxic activity against breast cancer cells. Another Portieria hornemannii sample was collected from Noordhoek in the Eastern Cape, it yielded one known compound referred to as 3Z-6-bromo-3-(bromomethylidene)-2,7- dichloro-7-methyloct-1-ene (3.5), as well as one new compound called portieric acid A (3.6) or 5-bromo-2-(bromomethylidene)-6-chloro-6-methylheptanoic acid. Portieric acid A showed slight cytotoxic activity and also represents a new class of compound within the genus Portieria. The isolation of secondary metabolites from the South African red alga, Laurencia glomerata, yielded two known compounds; 7-hydroxylaurene (4.9) and cis-neolaurencenyne (4.12), as well as one chamigrane related compound (4.11). Laurencia flexuosa yielded one known compound called 3Z-bromofucin (4.13). Using 1H NMR, GC and molecular systematics, a novel method for identifying different species of Laurencia was also investigated.
- Full Text:
The medicinal chemistry of Cyclo (D-PHE-4I-PRO) and Cyclo (L-PHE-4I-PRO)
- Authors: Qhola, Lipolelo
- Date: 2012
- Subjects: Cyclic peptides , Pharmaceutical chemistry , Peptide drugs
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10152 , http://hdl.handle.net/10948/d1011619 , Cyclic peptides , Pharmaceutical chemistry , Peptide drugs
- Description: Cyclic dipeptides have been widely used as pharmaceutical agents due to their favourable properties and the fact that they are more stable and membrane permeable than their linear analogues. These characteristics make cyclic dipeptides attractive to protein-based drug developers (Martins & Carvalho, 2007). In this research study, the method of Milne et al. (1992) was used to synthesize the protected linear dipeptide esters. This was followed by boiling the unprotected, linear dipeptide esters under reflux in an oil bath (Sec-butanol: toluene (4:1)). This method gave good yields and pure cyclic dipeptides. Scanning electron microscopy, thermal analysis, X-ray powder diffraction and differential scanning calorimetry were used for evaluation of the physiochemical properties of the cyclic dipeptides. High-performance liquid chromatography and thin layer chromatography were used to determine the purity of the cyclic dipeptides. The structures of the cyclic dipeptides were elucidated using infrared spectroscopy, mass spectrometry, nuclear magnetic resonance spectroscopy and molecular modeling and computational chemistry. The aim of the study was to determine the possible therapeutic activity of cyclo(D-Phe-4I-Pro) and cyclo(L-Phe-4I-Pro) with regard to antimicrobial, anticancer, antidiabetes and haematological effects. Both cyclic dipeptides showed a significant growth inhibition of Gram-positive, Gram-negative and fungal microorganisms in the antimicrobial study. Anticancer studies showed that both cyclic dipeptides caused growth inhibition of the MCF-7, HT-29 and HeLa cancer cell lines. Both cyclic dipeptides showed no antidiabetic activity. Haematological studies revealed that both cyclic dipeptides caused a significant effect on the clotting time and platelet aggregation. They caused an increase in clotting time and also inhibited platelet aggregation.
- Full Text:
- Authors: Qhola, Lipolelo
- Date: 2012
- Subjects: Cyclic peptides , Pharmaceutical chemistry , Peptide drugs
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10152 , http://hdl.handle.net/10948/d1011619 , Cyclic peptides , Pharmaceutical chemistry , Peptide drugs
- Description: Cyclic dipeptides have been widely used as pharmaceutical agents due to their favourable properties and the fact that they are more stable and membrane permeable than their linear analogues. These characteristics make cyclic dipeptides attractive to protein-based drug developers (Martins & Carvalho, 2007). In this research study, the method of Milne et al. (1992) was used to synthesize the protected linear dipeptide esters. This was followed by boiling the unprotected, linear dipeptide esters under reflux in an oil bath (Sec-butanol: toluene (4:1)). This method gave good yields and pure cyclic dipeptides. Scanning electron microscopy, thermal analysis, X-ray powder diffraction and differential scanning calorimetry were used for evaluation of the physiochemical properties of the cyclic dipeptides. High-performance liquid chromatography and thin layer chromatography were used to determine the purity of the cyclic dipeptides. The structures of the cyclic dipeptides were elucidated using infrared spectroscopy, mass spectrometry, nuclear magnetic resonance spectroscopy and molecular modeling and computational chemistry. The aim of the study was to determine the possible therapeutic activity of cyclo(D-Phe-4I-Pro) and cyclo(L-Phe-4I-Pro) with regard to antimicrobial, anticancer, antidiabetes and haematological effects. Both cyclic dipeptides showed a significant growth inhibition of Gram-positive, Gram-negative and fungal microorganisms in the antimicrobial study. Anticancer studies showed that both cyclic dipeptides caused growth inhibition of the MCF-7, HT-29 and HeLa cancer cell lines. Both cyclic dipeptides showed no antidiabetic activity. Haematological studies revealed that both cyclic dipeptides caused a significant effect on the clotting time and platelet aggregation. They caused an increase in clotting time and also inhibited platelet aggregation.
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The medicinal chemistry of cyclo(Phe-4CI-Pro) and Cyclo(D-Phe-4CI-Pro)
- Authors: Milne, Marnus
- Date: 2012
- Subjects: Cyclic peptides , Pharmaceutical chemistry , Peptide drugs
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10157 , http://hdl.handle.net/10948/d1011848 , Cyclic peptides , Pharmaceutical chemistry , Peptide drugs
- Description: Cyclic dipeptides have limited conformational freedom due to their diketopiperazine backbone and their small size. They are relatively simple to synthesise, making them ideal subjects for investigation into their biological effects. Cyclic dipeptides have also been known for their multitude of biological activities, including antimicrobial, anticancer and haematological properties. In this study the cyclic dipeptides, cyclo(Phe-4Cl-Pro) and cyclo(D-Phe-4Cl-Pro), were synthesised from their corresponding linear precursors using a modified phenol-induced cyclisation procedure. The phenol induced cyclisation procedure resulted in good yields and purity of the cyclic dipeptides. Quantitative analysis and evaluation of the physiochemical properties of the cyclic dipeptides was achieved using high-performance liquid chromatography, scanning electron microscopy, thermal analysis and X-ray powder diffraction. Structural elucidation of the cyclic dipeptides was done by means of infrared spectroscopy, mass spectroscopy, nuclear magnetic resonance spectroscopy and molecular modelling. The study‟s aim was to determine the biological activity of cyclo(Phe-4Cl-Pro) and cyclo(D-Phe-4Cl-Pro) with respect to their anticancer, antimicrobial, haematological and ant-diabetic studies. Anticancer studies revealed that cyclo(Phe-4Cl-Pro) and cyclo(D-Phe-4Cl-Pro) inhibited the growth of HeLa (cervical cancer), HT-29 (colon cancer) and MCF-7 (breast cancer) cancer cell lines. Both cyclic dipeptides also inhibited the growth of certain selected Gram-positive, Gram-negative and fungal microorganisms in the antimicrobial study. Although the inhibition of growth in the anticancer and antimicrobial studies was statistically significant, the clinical relevance is questionable, since the inhibition produced by both cyclic dipeptides was very limited compared to other pre-existing anticancer and antimicrobial agents. Both cyclic dipeptides caused a significant shortening of the APTT and PT clotting times and an increase in the fibrin and D-Dimer formation. Cyclo(D-Phe-4Cl-Pro) at a screening concentration of 12.5 mM and 3.125 mM, showed significant anti-platelet activity. Both cyclic dipeptides failed to produce any inhibition of the α-Glucosidase enzyme and very limited inhibition of the α-Amylase enzyme.
- Full Text:
- Authors: Milne, Marnus
- Date: 2012
- Subjects: Cyclic peptides , Pharmaceutical chemistry , Peptide drugs
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10157 , http://hdl.handle.net/10948/d1011848 , Cyclic peptides , Pharmaceutical chemistry , Peptide drugs
- Description: Cyclic dipeptides have limited conformational freedom due to their diketopiperazine backbone and their small size. They are relatively simple to synthesise, making them ideal subjects for investigation into their biological effects. Cyclic dipeptides have also been known for their multitude of biological activities, including antimicrobial, anticancer and haematological properties. In this study the cyclic dipeptides, cyclo(Phe-4Cl-Pro) and cyclo(D-Phe-4Cl-Pro), were synthesised from their corresponding linear precursors using a modified phenol-induced cyclisation procedure. The phenol induced cyclisation procedure resulted in good yields and purity of the cyclic dipeptides. Quantitative analysis and evaluation of the physiochemical properties of the cyclic dipeptides was achieved using high-performance liquid chromatography, scanning electron microscopy, thermal analysis and X-ray powder diffraction. Structural elucidation of the cyclic dipeptides was done by means of infrared spectroscopy, mass spectroscopy, nuclear magnetic resonance spectroscopy and molecular modelling. The study‟s aim was to determine the biological activity of cyclo(Phe-4Cl-Pro) and cyclo(D-Phe-4Cl-Pro) with respect to their anticancer, antimicrobial, haematological and ant-diabetic studies. Anticancer studies revealed that cyclo(Phe-4Cl-Pro) and cyclo(D-Phe-4Cl-Pro) inhibited the growth of HeLa (cervical cancer), HT-29 (colon cancer) and MCF-7 (breast cancer) cancer cell lines. Both cyclic dipeptides also inhibited the growth of certain selected Gram-positive, Gram-negative and fungal microorganisms in the antimicrobial study. Although the inhibition of growth in the anticancer and antimicrobial studies was statistically significant, the clinical relevance is questionable, since the inhibition produced by both cyclic dipeptides was very limited compared to other pre-existing anticancer and antimicrobial agents. Both cyclic dipeptides caused a significant shortening of the APTT and PT clotting times and an increase in the fibrin and D-Dimer formation. Cyclo(D-Phe-4Cl-Pro) at a screening concentration of 12.5 mM and 3.125 mM, showed significant anti-platelet activity. Both cyclic dipeptides failed to produce any inhibition of the α-Glucosidase enzyme and very limited inhibition of the α-Amylase enzyme.
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Synthesis of chromium carbene scaffolds for use in medicinal chemistry
- Rafael, Christopher Carlos Ferreira
- Authors: Rafael, Christopher Carlos Ferreira
- Date: 2014
- Subjects: Carbenes (Methylene compounds) , Pharmaceutical chemistry
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4456 , http://hdl.handle.net/10962/d1010863 , Carbenes (Methylene compounds) , Pharmaceutical chemistry
- Description: This study involves using methyllithium to synthesize Fischer carbene complexes as precursors for metal templated α,β-unsaturated complexes with potential as acceptors in the Baylis Hillman reaction as well as in Dötz benzannulation. Fischer carbene complexes contain low oxidation state metal centers, are electrophilic in nature and are stabilized by π-donating substituents such as alkoxy and amino groups. The increased electron withdrawing nature of the metal carbonyl moiety was expected to improve the rates of reaction compared to organic carbonyls. Four Fischer carbenes were synthesized via nucleophilic addition of MeLi to chromium and tungsten hexacarbonyl at low temperatures followed by alkylation using either a Meerwein salt (Me₃OBF₄) to give the desired Fischer metal methyl methoxy carbenes or Et₄NBr/alkylhalide to make the corresponding ethoxy and allyloxy carbenes. Characterization was by means of ¹³C NMR, ¹H NMR, and IR. In silico studies were carried out looking at the effect of substituents on the carbene bond. Synthesis of α,β-unsaturated complexes was effected via the aldol condensation route and found to be unfavorable using enolizable aldehydes, although the use of two aryl aldehydes resulted in successful preparation of two α,β-unsaturated complexes. Difficulty in the purification of these complexes hindered their full characterization. Computational studies looked at the effect of substituents on the system as well as variation of the metal from Cr to Mo and W. Synthesis of Baylis Hillman adducts using α,β-unsaturated complexes as acceptors was unsuccessful due to the ease of product oxidization. One potential product was obtained in its crude form although purification was not possible due to oxidation. Computational studies suggested that the oxygen on the ligand negatively impacts the stability of these Fischer carbene derived Baylis Hillman adducts promoting intramolecular oxidation of the metal. The α,β-unsaturated complexes and Baylis Hillman adducts were considered to be candidates to undergo Dötz benzannulation methodology. The use of the α,β-unsaturated complexes in this reaction was generally unsuccessful, both in the microwave and in conventional reflux conditions. Computational studies of these compounds were carried out to facilitate understanding of their stability and configuration.
- Full Text:
- Authors: Rafael, Christopher Carlos Ferreira
- Date: 2014
- Subjects: Carbenes (Methylene compounds) , Pharmaceutical chemistry
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4456 , http://hdl.handle.net/10962/d1010863 , Carbenes (Methylene compounds) , Pharmaceutical chemistry
- Description: This study involves using methyllithium to synthesize Fischer carbene complexes as precursors for metal templated α,β-unsaturated complexes with potential as acceptors in the Baylis Hillman reaction as well as in Dötz benzannulation. Fischer carbene complexes contain low oxidation state metal centers, are electrophilic in nature and are stabilized by π-donating substituents such as alkoxy and amino groups. The increased electron withdrawing nature of the metal carbonyl moiety was expected to improve the rates of reaction compared to organic carbonyls. Four Fischer carbenes were synthesized via nucleophilic addition of MeLi to chromium and tungsten hexacarbonyl at low temperatures followed by alkylation using either a Meerwein salt (Me₃OBF₄) to give the desired Fischer metal methyl methoxy carbenes or Et₄NBr/alkylhalide to make the corresponding ethoxy and allyloxy carbenes. Characterization was by means of ¹³C NMR, ¹H NMR, and IR. In silico studies were carried out looking at the effect of substituents on the carbene bond. Synthesis of α,β-unsaturated complexes was effected via the aldol condensation route and found to be unfavorable using enolizable aldehydes, although the use of two aryl aldehydes resulted in successful preparation of two α,β-unsaturated complexes. Difficulty in the purification of these complexes hindered their full characterization. Computational studies looked at the effect of substituents on the system as well as variation of the metal from Cr to Mo and W. Synthesis of Baylis Hillman adducts using α,β-unsaturated complexes as acceptors was unsuccessful due to the ease of product oxidization. One potential product was obtained in its crude form although purification was not possible due to oxidation. Computational studies suggested that the oxygen on the ligand negatively impacts the stability of these Fischer carbene derived Baylis Hillman adducts promoting intramolecular oxidation of the metal. The α,β-unsaturated complexes and Baylis Hillman adducts were considered to be candidates to undergo Dötz benzannulation methodology. The use of the α,β-unsaturated complexes in this reaction was generally unsuccessful, both in the microwave and in conventional reflux conditions. Computational studies of these compounds were carried out to facilitate understanding of their stability and configuration.
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Formulation and process optimisation of ethionamide 250 MGtablets using quality by design principles
- Date: 2015
- Subjects: Pharmaceutical chemistry , Drugs -- Design , Pharmaceutical technology
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/3979 , http://vital.seals.ac.za8080/10948/28876 , vital:20497
- Description: The traditional approach of Quality by Testing (QbT) limits the assurance of product quality to in-process and post-production testing. To overcome these limitations, a more proactive and systematic means to product development and optimisation is required. Quality by Design (QbD) is an example of such an approach which focuses on understanding the product and its manufacturing process and emphasises that quality should be built into the product and not merely tested. The study aims to optimise ethionamide tablets, an immediate release oral solid dosage form using QbD.
- Full Text:
Formulation and process optimisation of ethionamide 250 MGtablets using quality by design principles
- Date: 2015
- Subjects: Pharmaceutical chemistry , Drugs -- Design , Pharmaceutical technology
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/3979 , http://vital.seals.ac.za8080/10948/28876 , vital:20497
- Description: The traditional approach of Quality by Testing (QbT) limits the assurance of product quality to in-process and post-production testing. To overcome these limitations, a more proactive and systematic means to product development and optimisation is required. Quality by Design (QbD) is an example of such an approach which focuses on understanding the product and its manufacturing process and emphasises that quality should be built into the product and not merely tested. The study aims to optimise ethionamide tablets, an immediate release oral solid dosage form using QbD.
- Full Text:
Benzoyl isothiocyanates derived ligands as potential HIV-1 protease inhibitors and their reactions with gold ions
- Authors: Odame, Felix
- Date: 2016
- Subjects: HIV (Viruses) -- Enzymes , Enzyme inhibitors -- Research , Pharmaceutical chemistry , Biochemistry
- Language: English
- Type: Thesis , Doctoral , DPhil
- Identifier: http://hdl.handle.net/10948/33228 , vital:32585
- Description: The synthesis and evaluation of benzoyl isothiocyanate derivatives as potential HIV-1 protease inhibitors is presented. The ligands were first designed to fit the protease active site using Autodock 4.2. The design was based on the deNOVO method of drug design in which the active site coordinates from the crystal structure of protease bound to ritonavir was used. An attempt to access the scaffolds designed initially led to the formation of 2,2,4-trimethyl 2,3-dihydro-1H-1,5-benzodiazepin-5-ium isophthalate and 2-2-(3-methylphenyl-1Hbenzimidazole which could not be converted to the desired intermediate. A further attempt led to formation of amino acid and amino acid ester derivatives of benzoyl isothiocyanates which have been fully characterized and the reasons why the desired intermediates were not readily accessible explained. Scaffolds based on the benzoyl isothiocyanate derivatives of structurally diverse diamines were then screened. Sixty compounds have been synthesized and fully characterized using elemental analysis, spectroscopy, GC-MS and twenty-six crystal structures have been discussed. The DFT transition state studies of 11-phenyl- 1,8,10,12-tetraazatricyclo[7.4.0.02,7]trideca-2(7),3,5,9,11-pentaene-13-thione (20), N-(1Hbenzimidazol-2-yl)benzamide (21), 3-(1,3-benzothiazol-2-yl)-1-(benzoyl)thiourea (23), and N-[(9E)-8,10,17-triazatetracyclo[8.7.0.02,7.011,16]heptadeca-1(17),2,4,6,11(16),12,14-heptaen-9-ylidene] benzamide (39), have been carried out and their detailed density functional theory reaction mechanism have be computed. The Bernly algorithm was used in the determination of saddle points (transtions states), and the intrinsic reaction coordinates leading to the determination of intermediates were traced and optimized to a global minimum or in some cases a local minimum was obtained. The cell viability tests of diamine derivatives which was done by exposing white blood cells to the compounds (inhibitors) at 37 °C and a pH of 7.4 showed that 1-(4-bromobenzoyl)-3-[2- ({[(4-bromophenyl)formamido]methanethioyl}amino)phenyl]thiourea (46), 1-(3-chloro benzoyl)-3-[2-({[(3-chlorophenyl)formamido]methanethioyl}amino)phenyl]thiourea (48), 1- (3-bromobenzoyl)-3-[2-({[(3-bromophenyl)formamido]methanethioyl}amino)phenyl] thiourea (49) and 3-benzoyl-1-(4-{[(phenylformamido)methanethioyl]amino}butyl)thiourea (54), in that group of compounds were cytotoxic with EC50 values of 17.04 ± 9.75 μM, 69.20± 38.16 μM, 35.90 ± 20.55 μM and 68.37 ± 26.45 μM, respectively. 4-Bromo-N-[(9E)-8,10,17-triazatetracyclo[8.7.0.02,7.011,16]heptadeca-1(17),2,4,6,11(16),12,14-heptaen-9-ylidene] benzamide (32), 4-methoxy-N-[(9E)-8,10,17-triazatetracyclo[8.7.0.02,7.011,16]heptadeca-1(17),2,4,6,11(16),12,14-heptaen-9-ylidene]benzamide (33) and 3-chloro-N-[(9E)-8,10,17-triazatetracyclo[8.7.0.02,7.011,16]heptadeca-1(17),2,4,6,11(16),12,14-heptaen-9-ylidene] benzamide (37) were also cytotoxic giving EC50 values of 45.47 ± 21.92, 45.09 ±13.79 and 74.94 ± 13.17 μM, respectively. 3-(1,3-Benzothiazol-2-yl)-1-(3-bromobenzoyl)thiourea (31) and 3-(1,3-benzothiazoyl-2-yl)-1-(4-nitrobenzoyl)thiourea (30) derivatives were also found to be cytotoxic with EC50 values of 1.207 ± 0.58 and 24.08 ±13.14 nM, respectively. 11-(4-Chlorophenyl-1,8,10, 12-tetraazatricyclo[7.4.0.02,7]trideca-2(7),3,5,9,11-pentaene-13-thione (12), 11-(4-methoxyphenyl)-1,8,10,12-tetraazatricyclo[7.4.0.02,7]trideca-2(7),3,9,1-pentaene-13-thione (14), and 11-phenyl-1,8,10,12-tetraazatricyclo[7.4.0.02,7]trideca-2(7),3,5,9,11-pentaene-13-thione (20), were found to be cytotoxic giving EC50 values of 0.152 ± 0.051, 37.96 ± 21.87 and 5.28 ± 2.95 μM, respectively. In the enzyme inhibition studies compound 49 gave a percentage inhibition of 97.03 ± 10.61% at 100 μM, but the fact that it is cytoxic might make it less useful, whilst compounds 19 and 16 had a percentage inhibition of 59.57 ± 13.59% (4-nitro derivative) and 79.97 ± 11.97% (3-nitro derivative) respectively at 100 μM of inhibitor and 20 μM of enzyme (HIV-1 protease). The results suggests that the presence of the nitro group at position 3 (16) and 4 (19) leads to an increase in activity against HIV-1 protease.
- Full Text:
- Authors: Odame, Felix
- Date: 2016
- Subjects: HIV (Viruses) -- Enzymes , Enzyme inhibitors -- Research , Pharmaceutical chemistry , Biochemistry
- Language: English
- Type: Thesis , Doctoral , DPhil
- Identifier: http://hdl.handle.net/10948/33228 , vital:32585
- Description: The synthesis and evaluation of benzoyl isothiocyanate derivatives as potential HIV-1 protease inhibitors is presented. The ligands were first designed to fit the protease active site using Autodock 4.2. The design was based on the deNOVO method of drug design in which the active site coordinates from the crystal structure of protease bound to ritonavir was used. An attempt to access the scaffolds designed initially led to the formation of 2,2,4-trimethyl 2,3-dihydro-1H-1,5-benzodiazepin-5-ium isophthalate and 2-2-(3-methylphenyl-1Hbenzimidazole which could not be converted to the desired intermediate. A further attempt led to formation of amino acid and amino acid ester derivatives of benzoyl isothiocyanates which have been fully characterized and the reasons why the desired intermediates were not readily accessible explained. Scaffolds based on the benzoyl isothiocyanate derivatives of structurally diverse diamines were then screened. Sixty compounds have been synthesized and fully characterized using elemental analysis, spectroscopy, GC-MS and twenty-six crystal structures have been discussed. The DFT transition state studies of 11-phenyl- 1,8,10,12-tetraazatricyclo[7.4.0.02,7]trideca-2(7),3,5,9,11-pentaene-13-thione (20), N-(1Hbenzimidazol-2-yl)benzamide (21), 3-(1,3-benzothiazol-2-yl)-1-(benzoyl)thiourea (23), and N-[(9E)-8,10,17-triazatetracyclo[8.7.0.02,7.011,16]heptadeca-1(17),2,4,6,11(16),12,14-heptaen-9-ylidene] benzamide (39), have been carried out and their detailed density functional theory reaction mechanism have be computed. The Bernly algorithm was used in the determination of saddle points (transtions states), and the intrinsic reaction coordinates leading to the determination of intermediates were traced and optimized to a global minimum or in some cases a local minimum was obtained. The cell viability tests of diamine derivatives which was done by exposing white blood cells to the compounds (inhibitors) at 37 °C and a pH of 7.4 showed that 1-(4-bromobenzoyl)-3-[2- ({[(4-bromophenyl)formamido]methanethioyl}amino)phenyl]thiourea (46), 1-(3-chloro benzoyl)-3-[2-({[(3-chlorophenyl)formamido]methanethioyl}amino)phenyl]thiourea (48), 1- (3-bromobenzoyl)-3-[2-({[(3-bromophenyl)formamido]methanethioyl}amino)phenyl] thiourea (49) and 3-benzoyl-1-(4-{[(phenylformamido)methanethioyl]amino}butyl)thiourea (54), in that group of compounds were cytotoxic with EC50 values of 17.04 ± 9.75 μM, 69.20± 38.16 μM, 35.90 ± 20.55 μM and 68.37 ± 26.45 μM, respectively. 4-Bromo-N-[(9E)-8,10,17-triazatetracyclo[8.7.0.02,7.011,16]heptadeca-1(17),2,4,6,11(16),12,14-heptaen-9-ylidene] benzamide (32), 4-methoxy-N-[(9E)-8,10,17-triazatetracyclo[8.7.0.02,7.011,16]heptadeca-1(17),2,4,6,11(16),12,14-heptaen-9-ylidene]benzamide (33) and 3-chloro-N-[(9E)-8,10,17-triazatetracyclo[8.7.0.02,7.011,16]heptadeca-1(17),2,4,6,11(16),12,14-heptaen-9-ylidene] benzamide (37) were also cytotoxic giving EC50 values of 45.47 ± 21.92, 45.09 ±13.79 and 74.94 ± 13.17 μM, respectively. 3-(1,3-Benzothiazol-2-yl)-1-(3-bromobenzoyl)thiourea (31) and 3-(1,3-benzothiazoyl-2-yl)-1-(4-nitrobenzoyl)thiourea (30) derivatives were also found to be cytotoxic with EC50 values of 1.207 ± 0.58 and 24.08 ±13.14 nM, respectively. 11-(4-Chlorophenyl-1,8,10, 12-tetraazatricyclo[7.4.0.02,7]trideca-2(7),3,5,9,11-pentaene-13-thione (12), 11-(4-methoxyphenyl)-1,8,10,12-tetraazatricyclo[7.4.0.02,7]trideca-2(7),3,9,1-pentaene-13-thione (14), and 11-phenyl-1,8,10,12-tetraazatricyclo[7.4.0.02,7]trideca-2(7),3,5,9,11-pentaene-13-thione (20), were found to be cytotoxic giving EC50 values of 0.152 ± 0.051, 37.96 ± 21.87 and 5.28 ± 2.95 μM, respectively. In the enzyme inhibition studies compound 49 gave a percentage inhibition of 97.03 ± 10.61% at 100 μM, but the fact that it is cytoxic might make it less useful, whilst compounds 19 and 16 had a percentage inhibition of 59.57 ± 13.59% (4-nitro derivative) and 79.97 ± 11.97% (3-nitro derivative) respectively at 100 μM of inhibitor and 20 μM of enzyme (HIV-1 protease). The results suggests that the presence of the nitro group at position 3 (16) and 4 (19) leads to an increase in activity against HIV-1 protease.
- Full Text:
The development and evaluation of a new manufacturing process for β-sitosterol-D glucoside
- Mtyopo, Mthetheleli Bethwell
- Authors: Mtyopo, Mthetheleli Bethwell
- Date: 2016
- Subjects: Pharmaceutical chemistry , Chemistry, Organic
- Language: English
- Type: Thesis , Doctoral , DTech
- Identifier: http://hdl.handle.net/10948/45920 , vital:39320
- Description: The existing production sequence of β-sitosterol-D-glucoside, a glucoside used in an “over-the-counter” (OTC) preparation under the brand name of Moducare® comprises of three process steps with an overall yield of less than 20%. The low yield is partly due to the instability of intermediates at reaction temperatures > 0oC, and partly due to the thermodynamic equilibrium between two stereoisomers. An economically alternative process was developed, evaluated and scaled-up in a 2l reactor. The project was initiated with a specific limitation in terms of the starting material that comprised a mixture of plant sterols, which necessitated a study of the isolation and purification of the desired product from a rather complex reaction mixture. The use of silver as halide acceptor for the Koenigs-Knorr synthesis did not give statistically significant different results from the same approach but using cadmium as halide acceptor instead. However, using the direct O-glucosylation approach not only gave statistically significant higher results, but also resulted in a much more convenient procedure. Under optimum conditions, a yield of approximately 83% (isolated) of 2,3,4,6- tetra-О-acetyl-β-sitosterol-D-glucoside could be achieved, which was substantially higher than that achieved with the traditional Koenigs-Knorr methodology and above reported yields in the literature (60-80%) for direct glycosylation. Separation of 2,3,4,6-tetra-О-acetyl-β-sitosterol-D-glucoside (BSSGT) from a reaction mixture that contains 2,3,4,6-tetra-О-acetyl-campesterol-D-glucoside (CSGT), 2,3,4,6-tetra-О-acetyl campestanol-glucoside (CSSGT), and 2,3,4,6-tetra-О-acetyl-sitostanol-Dglucoside (SSGT) was investigated using column chromatography. When using silica gel particles, very good separation efficiency and product recovery could be achieved using hexane/ethyl hexane as eluent. The isolated 2,3,4,6-tetra-О-acetyl-β-sitosterol-Dglucoside was easily hydrolysed to β-sitosterol-D-glucoside in high yields (79%) using methanolic KOH. The process for the production of β-sitosterol-D-glucoside was scaled-up from the laboratory bench scale (250 cm3) to a laboratory scale of 2 l using the direct Oglycosylation method. The overall yields of the scaled reaction for β-sitosterol-D-glucoside was slightly above the literature reported values (59%, 8/92) for the KnoenigsKnorr synthesis and compares well above (62%, 0/100) the current production process (less than 20% yields). When using catalogue prices, the material costs (without recycling) for the direct Oglucosylation route is approximately 57% less for the synthesis of 1kg of β-sitosterol-Dglucoside compared to the Koenigs-Knorr route. Given further savings for recycling, the direct O-glucosylation route provides an attractive alternative route for the synthesis ofthe target compound.
- Full Text:
- Authors: Mtyopo, Mthetheleli Bethwell
- Date: 2016
- Subjects: Pharmaceutical chemistry , Chemistry, Organic
- Language: English
- Type: Thesis , Doctoral , DTech
- Identifier: http://hdl.handle.net/10948/45920 , vital:39320
- Description: The existing production sequence of β-sitosterol-D-glucoside, a glucoside used in an “over-the-counter” (OTC) preparation under the brand name of Moducare® comprises of three process steps with an overall yield of less than 20%. The low yield is partly due to the instability of intermediates at reaction temperatures > 0oC, and partly due to the thermodynamic equilibrium between two stereoisomers. An economically alternative process was developed, evaluated and scaled-up in a 2l reactor. The project was initiated with a specific limitation in terms of the starting material that comprised a mixture of plant sterols, which necessitated a study of the isolation and purification of the desired product from a rather complex reaction mixture. The use of silver as halide acceptor for the Koenigs-Knorr synthesis did not give statistically significant different results from the same approach but using cadmium as halide acceptor instead. However, using the direct O-glucosylation approach not only gave statistically significant higher results, but also resulted in a much more convenient procedure. Under optimum conditions, a yield of approximately 83% (isolated) of 2,3,4,6- tetra-О-acetyl-β-sitosterol-D-glucoside could be achieved, which was substantially higher than that achieved with the traditional Koenigs-Knorr methodology and above reported yields in the literature (60-80%) for direct glycosylation. Separation of 2,3,4,6-tetra-О-acetyl-β-sitosterol-D-glucoside (BSSGT) from a reaction mixture that contains 2,3,4,6-tetra-О-acetyl-campesterol-D-glucoside (CSGT), 2,3,4,6-tetra-О-acetyl campestanol-glucoside (CSSGT), and 2,3,4,6-tetra-О-acetyl-sitostanol-Dglucoside (SSGT) was investigated using column chromatography. When using silica gel particles, very good separation efficiency and product recovery could be achieved using hexane/ethyl hexane as eluent. The isolated 2,3,4,6-tetra-О-acetyl-β-sitosterol-Dglucoside was easily hydrolysed to β-sitosterol-D-glucoside in high yields (79%) using methanolic KOH. The process for the production of β-sitosterol-D-glucoside was scaled-up from the laboratory bench scale (250 cm3) to a laboratory scale of 2 l using the direct Oglycosylation method. The overall yields of the scaled reaction for β-sitosterol-D-glucoside was slightly above the literature reported values (59%, 8/92) for the KnoenigsKnorr synthesis and compares well above (62%, 0/100) the current production process (less than 20% yields). When using catalogue prices, the material costs (without recycling) for the direct Oglucosylation route is approximately 57% less for the synthesis of 1kg of β-sitosterol-Dglucoside compared to the Koenigs-Knorr route. Given further savings for recycling, the direct O-glucosylation route provides an attractive alternative route for the synthesis ofthe target compound.
- Full Text:
Synthesis of L-menthyl glyoxylate, an important intermediate in the manufacture of ARVS, using flow chemistry technology
- Date: 2017
- Subjects: Chemistry , Pharmaceutical chemistry , Organic compounds -- Synthesis
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/12019 , http://vital.seals.ac.za8080/10948/29047 , vital:27018
- Description: Herein an alternative approach to the conventional batch synthesis of L-menthyl glyoxylate hydrate (MGH), an important intermediate in the synthesis of drugs of importance is reported, through flow chemistry technology. MGH was initially synthesized in batch and various reaction parameters optimized. It was found to proceed to completion after 6 hours of esterifying glyoxylic acid with excess alcohol (L-menthol) in the presence of a catalyst, ideally amberlyst-15 (an ion exchange resin) at 105 °C giving a yield of 72 %. The batch reaction conditions were adopted in a continuous flow synthesis setup, using the Labtrix Start system, in which reaction conditions were optimized. The optimization of glyoxylic acid conversion (92 %) in the Labtrix Start system gave reaction conditions that resulted in low MGH selectivity (25 %) whereas the optimization for MGH selectivity (100 %) gave a conversion a poor glyoxylic acid conversion (15 %). The FlowSyn system fitted with a column reactor gave the best results, in which the optimum conditions were an excess of L-menthol (1.5 M, 6.0 equiv.), temperature (80 °C) and a residence time of 2.5 minutes with a high selectivity (77 %) and average conversion (50 %). The optimized reaction conditions for conversion and selectivity on the different flow systems did not vary significantly and similar trends were observed for the systems. It was shown that an increase in temperature, mole equivalents and residence time led to an increase in MGH conversion in all flow systems. The scale up of the esterification reaction from the Labtrix Start system (19 μL microreactor) to the FlowSyn system fitted with a 2 mL reactor chip, showed that the reaction proceeds with a slight drop in selectivity from 100 % to 92 % while conversion dropped from 15 to 12 %. On the contrary, a significant drop in conversion and selectivity were observed when the FlowSyn column reactor was up-scaled to the Elite-tubular furnace, owing to the poor mixing in the larger channel size reactor.
- Full Text:
- Date: 2017
- Subjects: Chemistry , Pharmaceutical chemistry , Organic compounds -- Synthesis
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/12019 , http://vital.seals.ac.za8080/10948/29047 , vital:27018
- Description: Herein an alternative approach to the conventional batch synthesis of L-menthyl glyoxylate hydrate (MGH), an important intermediate in the synthesis of drugs of importance is reported, through flow chemistry technology. MGH was initially synthesized in batch and various reaction parameters optimized. It was found to proceed to completion after 6 hours of esterifying glyoxylic acid with excess alcohol (L-menthol) in the presence of a catalyst, ideally amberlyst-15 (an ion exchange resin) at 105 °C giving a yield of 72 %. The batch reaction conditions were adopted in a continuous flow synthesis setup, using the Labtrix Start system, in which reaction conditions were optimized. The optimization of glyoxylic acid conversion (92 %) in the Labtrix Start system gave reaction conditions that resulted in low MGH selectivity (25 %) whereas the optimization for MGH selectivity (100 %) gave a conversion a poor glyoxylic acid conversion (15 %). The FlowSyn system fitted with a column reactor gave the best results, in which the optimum conditions were an excess of L-menthol (1.5 M, 6.0 equiv.), temperature (80 °C) and a residence time of 2.5 minutes with a high selectivity (77 %) and average conversion (50 %). The optimized reaction conditions for conversion and selectivity on the different flow systems did not vary significantly and similar trends were observed for the systems. It was shown that an increase in temperature, mole equivalents and residence time led to an increase in MGH conversion in all flow systems. The scale up of the esterification reaction from the Labtrix Start system (19 μL microreactor) to the FlowSyn system fitted with a 2 mL reactor chip, showed that the reaction proceeds with a slight drop in selectivity from 100 % to 92 % while conversion dropped from 15 to 12 %. On the contrary, a significant drop in conversion and selectivity were observed when the FlowSyn column reactor was up-scaled to the Elite-tubular furnace, owing to the poor mixing in the larger channel size reactor.
- Full Text:
Application of a quality by design approach to optimise an existing product
- Authors: Maxwell, Taryn Lee
- Date: 2018
- Subjects: Pharmaceutical chemistry , Drugs -- Design , Pharmaceutical technology
- Language: English
- Type: Thesis , Masters , MPharm
- Identifier: http://hdl.handle.net/10948/32752 , vital:32341
- Description: Quality by design is a science and risk based approach whereby quality is built into the product or process during the pharmaceutical development. although quality by design is encouraged for pharmaceutical development. it is possible to apply quality by design to optimize an existing product as part of a continual improvement strategy. the purpose of this study is to determine which factors should be considered to justify the application of quality by design to optimize an existing product.
- Full Text: false
- Authors: Maxwell, Taryn Lee
- Date: 2018
- Subjects: Pharmaceutical chemistry , Drugs -- Design , Pharmaceutical technology
- Language: English
- Type: Thesis , Masters , MPharm
- Identifier: http://hdl.handle.net/10948/32752 , vital:32341
- Description: Quality by design is a science and risk based approach whereby quality is built into the product or process during the pharmaceutical development. although quality by design is encouraged for pharmaceutical development. it is possible to apply quality by design to optimize an existing product as part of a continual improvement strategy. the purpose of this study is to determine which factors should be considered to justify the application of quality by design to optimize an existing product.
- Full Text: false
Optimisation of a legacy product with a history of tablet friability failures utilising quality by design
- Authors: Watkins, Eric
- Date: 2018
- Subjects: Pharmaceutical chemistry , Pharmaceutical industry , Pharmacy -- Research
- Language: English
- Type: Thesis , Masters , Degree
- Identifier: http://hdl.handle.net/10948/36398 , vital:33938
- Description: The concept of Quality by Design (QbD) was introduced as a method of building quality into the product during the initial stages of manufacturing. This study explores the suitability of utilising QbD to optimise a legacy product. With the aid of QbD, a higher level of quality assurance and product knowledge was achieved. Sound scientific and risk-based decisions allowed for a robust manufacturing process with inherent operational quality and flexibility. By the establishment a quality target product profile (QTPP) and determining the influence of the critical processing parameters (CPP's) on the product's critical quality attributes (cQA's) the process understanding of Product X can be more accurately defined. The relationships between several explanatory variables will be explored by using a sequence of Design of Experiments (DoE) to obtain an optimal response. The DoE were performed and analysed using Minitab® statistical software version 17.0 (Minitab Inc., United Kingdom). A Response Surface Methodology (RSM) using a central composite experimental design (CCD) was utilised to capture the data. The data was analysed using the collection of statistical models (ANOVA) to analyse the differences between the means and their associated procedures. Input variables investigated were: compression machine tooling shape, hardness, and loss on drying LOD (post drying). The significant value (α) of 0.05 helped to determine if the null hypothesis would be accepted or rejected. The DoE identified the factors that had the highest risk of affecting the output variables and helped to establish the design space. Post completion of the DoE, a confirmatory batch was made which served as a diagnostic tool for evaluating the effectiveness of the generated model. The establishment of a strategy to control the variables and responses is of critical importance in order to appropriately use the flexibility given to products developed or optimised using QbD principles. This study show that the structured approach used in Quality by Design methodology can be successfully applied to optimise a commercialised legacy product.
- Full Text:
- Authors: Watkins, Eric
- Date: 2018
- Subjects: Pharmaceutical chemistry , Pharmaceutical industry , Pharmacy -- Research
- Language: English
- Type: Thesis , Masters , Degree
- Identifier: http://hdl.handle.net/10948/36398 , vital:33938
- Description: The concept of Quality by Design (QbD) was introduced as a method of building quality into the product during the initial stages of manufacturing. This study explores the suitability of utilising QbD to optimise a legacy product. With the aid of QbD, a higher level of quality assurance and product knowledge was achieved. Sound scientific and risk-based decisions allowed for a robust manufacturing process with inherent operational quality and flexibility. By the establishment a quality target product profile (QTPP) and determining the influence of the critical processing parameters (CPP's) on the product's critical quality attributes (cQA's) the process understanding of Product X can be more accurately defined. The relationships between several explanatory variables will be explored by using a sequence of Design of Experiments (DoE) to obtain an optimal response. The DoE were performed and analysed using Minitab® statistical software version 17.0 (Minitab Inc., United Kingdom). A Response Surface Methodology (RSM) using a central composite experimental design (CCD) was utilised to capture the data. The data was analysed using the collection of statistical models (ANOVA) to analyse the differences between the means and their associated procedures. Input variables investigated were: compression machine tooling shape, hardness, and loss on drying LOD (post drying). The significant value (α) of 0.05 helped to determine if the null hypothesis would be accepted or rejected. The DoE identified the factors that had the highest risk of affecting the output variables and helped to establish the design space. Post completion of the DoE, a confirmatory batch was made which served as a diagnostic tool for evaluating the effectiveness of the generated model. The establishment of a strategy to control the variables and responses is of critical importance in order to appropriately use the flexibility given to products developed or optimised using QbD principles. This study show that the structured approach used in Quality by Design methodology can be successfully applied to optimise a commercialised legacy product.
- Full Text:
New synergic biomaterials for anti-cancer therapy
- Authors: Swanepoel, Bresler
- Date: 2019
- Subjects: Pharmaceutical chemistry , Cancer -- Research , Biomedical materials
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/43957 , vital:37087
- Description: In the last two decades, anti-cancer therapy has grown considerably with the help of both natural and synthetic anti-cancer compounds but, the search for new and improved cancer treatment remains an ongoing and important issue. Some anti-cancer compounds such as cisplatin are limited by their toxicity in normal tissues and the development of drug resistance. Therefore, in order to address drug resistance and side-effects of anti-cancer agents, recent research has been focusing on finding novel combinations of anti-cancer agents that have non-overlapping mechanisms of action. The first objective of this study was to determine the mechanism of action of Anemone nemorosa, Artemisia afra, N-[[3-(4-bromophenyl)-1H-pyrazol-5-yl]-carbamothioyl]-4-chloro-benzamide (BC-7) and N-benzoyl-N’-(3-(4-bromophenyl)-1H-pyrazol-5-yl)-thiourea (BT-7) through cell cycle arrest, phosphatidylserine translocation (PS), caspase activation and mitochondrial membrane depolarization. This study has shown that A. nemorosa, BC-7 and A. afra are capable of inducing cell death within three cancer cell lines namely HeLa, MeWo and HepG2, at varying degrees. HeLa cells were the most susceptible to treatment with A. nemorosa and BC-7 with IC50 values of 20.33 ± 2.480 μg/ml and 65.58 ± 8.400 μM (28.58 ± 3.660 μg/ml), respectively. A. afra was the most active against HepG2 cells with an IC50 value of 37.55 μg/ml. BT-7 was not cytotoxic against any of the cancer cell lines. The effects on HeLa cells and their progression through the cell cycle indicated that cells were arrested in the early M phase for all treatments. The induction of apoptosis was confirmed by an increase in PS translocation and activation of caspase 3 and 8 as well as a decrease in the mitochondrial membrane potential. It was deduced that A. nemorosa, A. afra and BC-7 induce caspase-dependent apoptosis in a mitochondrial dependent manner. The second objective of this study was to investigate the potential of A. nemorosa, A. afra and BC-7 to target various mediators involved in the inflammatory response as an alternative method in which cell death may be induced. Most treatments indicated that a tumour-elicited inflammatory response is indeed induced in HeLa cells and that the significant activation of nuclear factor kappa B (NF-κB) favoured the production of nitric oxide (NO) over cyclo-oxygenase 2 (COX-2). However, treatments with A. nemorosa, BC-7 and A. afra at their IC10 showed the potential of inhibiting this response. ROS levels were increased by most treatments and support the idea of ROS-mediated apoptosis. The third objective was to investigate combination treatments of these extracts and compounds for their potential synergistic cytotoxic activity and thus formulating the combinations as potential anti-cancer agents. Thirty combination mixtures were prepared using the IC50 values of each extract or compound at ratios of 1:3, 1:2, 1:1, 2:1 and 3:1, respectively. The cytotoxic/anti-proliferative activity of each mixture was determined by the bisBenzamide H 33342 trihydrochloride/propidium iodide (Hoechst 33342/PI) dual staining method on HeLa cervical cancer cells. The combination index (CI) values, at inhibition of 50% of HeLa cell growth, for each combination mixture, were determined by means of the Chou and Talalay method. The combined effect can then be indicated as CI < 1, synergism; CI = 1, additive effect or CI > 1, antagonism, respectively. Most combination treatments showed to have an antagonistic effect except for cisplatin:BC-7 (1:3, 1:1, 2:1, 3:1) and cisplatin:A. afra (1:3, 1:2, 1:1, 3:1) combinations that showed synergism. The 1:2 ratio of cisplatin:BC-7 and the 2:1 ratio of cisplatin:A. afra were additive. CI values were also calculated at inhibition of 10, 25 and 75% of HeLa cell growth, for each combination mixture. Antagonistic effects were frequently observed at lower effect levels such as at 10 and 25% inhibition of growth. However, this was not seen for the cisplatin:BC-7 combinations as all the ratios indicated synergism. Some of these ratios, such as the 1:3 and 1:2, even led to a greater degree of synergism being obtained, with noticeable antagonistic effects seen at 50 and 75% inhibition of growth. The current finding is that BC-7 and A. afra could lower the dose of cisplatin in combination to achieve a similar anti-cancer efficacy compared to the higher cisplatin dose when used alone. The lower dosage in combination could result in reduced drug resistance as well as limit the toxicity on normal cells associated with cisplatin treatment. In conclusion, this study shows, for the first time, that A. nemorosa has the potential to induce apoptosis and also has some anti- and pro-inflammatory activity in HeLa cancer cells. This study also enhanced the knowledge of the mechanism of apoptosis induction of BC-7, in a more detailed manner, as well as investigated its inflammatory effects for the first time. Results obtained for A. afra correlated nicely to previously reported studies and confirmed that the methods used in this study, although different, leads to the same conclusions. Combination treatments also indicated, for the first time, that BC-7 and A. afra have the ability to function in a synergic manner with cisplatin and proves that, although extensive research may have been done on a plant or compound, more can be discovered. This new information can lead to identification of new compounds in the plants and the integration of signalling pathways that can be targeted for treatment of cancer.
- Full Text:
- Authors: Swanepoel, Bresler
- Date: 2019
- Subjects: Pharmaceutical chemistry , Cancer -- Research , Biomedical materials
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/43957 , vital:37087
- Description: In the last two decades, anti-cancer therapy has grown considerably with the help of both natural and synthetic anti-cancer compounds but, the search for new and improved cancer treatment remains an ongoing and important issue. Some anti-cancer compounds such as cisplatin are limited by their toxicity in normal tissues and the development of drug resistance. Therefore, in order to address drug resistance and side-effects of anti-cancer agents, recent research has been focusing on finding novel combinations of anti-cancer agents that have non-overlapping mechanisms of action. The first objective of this study was to determine the mechanism of action of Anemone nemorosa, Artemisia afra, N-[[3-(4-bromophenyl)-1H-pyrazol-5-yl]-carbamothioyl]-4-chloro-benzamide (BC-7) and N-benzoyl-N’-(3-(4-bromophenyl)-1H-pyrazol-5-yl)-thiourea (BT-7) through cell cycle arrest, phosphatidylserine translocation (PS), caspase activation and mitochondrial membrane depolarization. This study has shown that A. nemorosa, BC-7 and A. afra are capable of inducing cell death within three cancer cell lines namely HeLa, MeWo and HepG2, at varying degrees. HeLa cells were the most susceptible to treatment with A. nemorosa and BC-7 with IC50 values of 20.33 ± 2.480 μg/ml and 65.58 ± 8.400 μM (28.58 ± 3.660 μg/ml), respectively. A. afra was the most active against HepG2 cells with an IC50 value of 37.55 μg/ml. BT-7 was not cytotoxic against any of the cancer cell lines. The effects on HeLa cells and their progression through the cell cycle indicated that cells were arrested in the early M phase for all treatments. The induction of apoptosis was confirmed by an increase in PS translocation and activation of caspase 3 and 8 as well as a decrease in the mitochondrial membrane potential. It was deduced that A. nemorosa, A. afra and BC-7 induce caspase-dependent apoptosis in a mitochondrial dependent manner. The second objective of this study was to investigate the potential of A. nemorosa, A. afra and BC-7 to target various mediators involved in the inflammatory response as an alternative method in which cell death may be induced. Most treatments indicated that a tumour-elicited inflammatory response is indeed induced in HeLa cells and that the significant activation of nuclear factor kappa B (NF-κB) favoured the production of nitric oxide (NO) over cyclo-oxygenase 2 (COX-2). However, treatments with A. nemorosa, BC-7 and A. afra at their IC10 showed the potential of inhibiting this response. ROS levels were increased by most treatments and support the idea of ROS-mediated apoptosis. The third objective was to investigate combination treatments of these extracts and compounds for their potential synergistic cytotoxic activity and thus formulating the combinations as potential anti-cancer agents. Thirty combination mixtures were prepared using the IC50 values of each extract or compound at ratios of 1:3, 1:2, 1:1, 2:1 and 3:1, respectively. The cytotoxic/anti-proliferative activity of each mixture was determined by the bisBenzamide H 33342 trihydrochloride/propidium iodide (Hoechst 33342/PI) dual staining method on HeLa cervical cancer cells. The combination index (CI) values, at inhibition of 50% of HeLa cell growth, for each combination mixture, were determined by means of the Chou and Talalay method. The combined effect can then be indicated as CI < 1, synergism; CI = 1, additive effect or CI > 1, antagonism, respectively. Most combination treatments showed to have an antagonistic effect except for cisplatin:BC-7 (1:3, 1:1, 2:1, 3:1) and cisplatin:A. afra (1:3, 1:2, 1:1, 3:1) combinations that showed synergism. The 1:2 ratio of cisplatin:BC-7 and the 2:1 ratio of cisplatin:A. afra were additive. CI values were also calculated at inhibition of 10, 25 and 75% of HeLa cell growth, for each combination mixture. Antagonistic effects were frequently observed at lower effect levels such as at 10 and 25% inhibition of growth. However, this was not seen for the cisplatin:BC-7 combinations as all the ratios indicated synergism. Some of these ratios, such as the 1:3 and 1:2, even led to a greater degree of synergism being obtained, with noticeable antagonistic effects seen at 50 and 75% inhibition of growth. The current finding is that BC-7 and A. afra could lower the dose of cisplatin in combination to achieve a similar anti-cancer efficacy compared to the higher cisplatin dose when used alone. The lower dosage in combination could result in reduced drug resistance as well as limit the toxicity on normal cells associated with cisplatin treatment. In conclusion, this study shows, for the first time, that A. nemorosa has the potential to induce apoptosis and also has some anti- and pro-inflammatory activity in HeLa cancer cells. This study also enhanced the knowledge of the mechanism of apoptosis induction of BC-7, in a more detailed manner, as well as investigated its inflammatory effects for the first time. Results obtained for A. afra correlated nicely to previously reported studies and confirmed that the methods used in this study, although different, leads to the same conclusions. Combination treatments also indicated, for the first time, that BC-7 and A. afra have the ability to function in a synergic manner with cisplatin and proves that, although extensive research may have been done on a plant or compound, more can be discovered. This new information can lead to identification of new compounds in the plants and the integration of signalling pathways that can be targeted for treatment of cancer.
- Full Text:
Statistical optimisation of a terbinafine-containing cream
- Authors: Strydom, Lana
- Date: 2019
- Subjects: Chemicals -- Physiological effect , Pharmaceutical chemistry , Medical microbiology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/44067 , vital:37101
- Description: Terbinafine hydrochloride (TBH) belongs to the allylamine class of antifungals and displays a favourable dermatopharmacokinetic profile, being both lipophilic and keratinophilic. It has thus been included in a variety of topical dosage forms for the treatment of dermatomycoses, many of which have been the subject of optimisation studies, with the purpose of improving the product. Since a TBH-containing cream had not been found in literature to have been optimised before, the aim of this study was to optimise a TBH cream formulation. A TBH cream formulation, suitable for optimisation, was developed. Preformulation tests were undertaken, including active-excipient compatibility testing using a combined thermal method consisting of both differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA), as well as isothermal stress testing (IST). With the confirmation of the suitability of the selected excipients, development of a suitable TBH cream took place largely by a trial-and-error approach. The choice of a suitable formulation was made based on the physical appearance and viscosity of the cream, for which a viscosity specification was set. The identified TBH cream formulation was evaluated for its physical appearance and physicochemical aspects to confirm its appropriateness for the purpose of further optimisation. Based on literature and observations from the preformulation and formulation stage, factors and responses for study during the optimisation stage were identified to study using a circumscribed central composite design (CCCD) at five levels (-1.612, -1, 0, +1, +1.612). Total percentage of surfactant (TPS), homogenisation speed (HS) and cooling rate (CR), were selected as factors to study their influence on cream viscosity, in vitro TBH release from the cream, as well as the chemical stability of TBH within the cream formulation. Following the application of stepwise multiple linear regression to the mathematical models, a suitable prediction model was only obtained for one response, cream viscosity at a shear rate of 20 s-1. A linear model was also found to fit the data for % in vitro TBH release after one hour, although a low R2 of 0.497 made the model unsuitable for prediction purposes. No mathematical model could be fit to the results for the response assessing the change in TBH concentration following seven days’ storage at accelerated stability conditions. The determination of the optimum TBH cream formulation was made chiefly on the basis of the results for cream viscosity and the optimised formulation was identified to have a predicted viscosity of 8.33 Pa.s and the factor settings to obtain this cream were a CR of 1.3 °C/min, HS set at 3400 rpm, and TPS of 4.2 %. Validation of the optimised TBH cream formulation was performed for cream viscosity at a shear rate of 20 s-1 and revealed that there was good agreement between the measured and predicted viscosity values. The optimised TBH cream underwent rheological characterisation and was compared to the innovator, Lamisil® cream, with both creams found to meet the desirable rheological profile. In vitro release testing (IVRT) was used to compare the release of TBH from the optimised TBH cream and Lamisil® cream, and the optimised TBH cream was found to show much greater TBH release over a six-hour time period than Lamisil® cream. Stability testing of the optimised TBH cream took place at accelerated stability testing conditions of 40 °C ± 2 °C and 75 ± 5 % relative humidity (RH). To assay the formulation and determine content uniformity over the three-month storage period, a suitable stability-indicating reversed-phase high performance liquid chromatography (RP-HPLC) method was developed and validated. Other cream properties which were tested included: qualitative aspects, viscosity, pH and microbial limits. At the end of the three-month stability testing period, the cream was found to meet most of the specifications set, except for cream homogeneity and viscosity. A TBH cream formulation was thus developed and optimised to meet a certain viscosity specification. Although this formulation was found to meet the viscosity specification on the day after its manufacture, the viscosity was found to increase on storage, such that it was outside the set viscosity specification range.
- Full Text:
- Authors: Strydom, Lana
- Date: 2019
- Subjects: Chemicals -- Physiological effect , Pharmaceutical chemistry , Medical microbiology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/44067 , vital:37101
- Description: Terbinafine hydrochloride (TBH) belongs to the allylamine class of antifungals and displays a favourable dermatopharmacokinetic profile, being both lipophilic and keratinophilic. It has thus been included in a variety of topical dosage forms for the treatment of dermatomycoses, many of which have been the subject of optimisation studies, with the purpose of improving the product. Since a TBH-containing cream had not been found in literature to have been optimised before, the aim of this study was to optimise a TBH cream formulation. A TBH cream formulation, suitable for optimisation, was developed. Preformulation tests were undertaken, including active-excipient compatibility testing using a combined thermal method consisting of both differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA), as well as isothermal stress testing (IST). With the confirmation of the suitability of the selected excipients, development of a suitable TBH cream took place largely by a trial-and-error approach. The choice of a suitable formulation was made based on the physical appearance and viscosity of the cream, for which a viscosity specification was set. The identified TBH cream formulation was evaluated for its physical appearance and physicochemical aspects to confirm its appropriateness for the purpose of further optimisation. Based on literature and observations from the preformulation and formulation stage, factors and responses for study during the optimisation stage were identified to study using a circumscribed central composite design (CCCD) at five levels (-1.612, -1, 0, +1, +1.612). Total percentage of surfactant (TPS), homogenisation speed (HS) and cooling rate (CR), were selected as factors to study their influence on cream viscosity, in vitro TBH release from the cream, as well as the chemical stability of TBH within the cream formulation. Following the application of stepwise multiple linear regression to the mathematical models, a suitable prediction model was only obtained for one response, cream viscosity at a shear rate of 20 s-1. A linear model was also found to fit the data for % in vitro TBH release after one hour, although a low R2 of 0.497 made the model unsuitable for prediction purposes. No mathematical model could be fit to the results for the response assessing the change in TBH concentration following seven days’ storage at accelerated stability conditions. The determination of the optimum TBH cream formulation was made chiefly on the basis of the results for cream viscosity and the optimised formulation was identified to have a predicted viscosity of 8.33 Pa.s and the factor settings to obtain this cream were a CR of 1.3 °C/min, HS set at 3400 rpm, and TPS of 4.2 %. Validation of the optimised TBH cream formulation was performed for cream viscosity at a shear rate of 20 s-1 and revealed that there was good agreement between the measured and predicted viscosity values. The optimised TBH cream underwent rheological characterisation and was compared to the innovator, Lamisil® cream, with both creams found to meet the desirable rheological profile. In vitro release testing (IVRT) was used to compare the release of TBH from the optimised TBH cream and Lamisil® cream, and the optimised TBH cream was found to show much greater TBH release over a six-hour time period than Lamisil® cream. Stability testing of the optimised TBH cream took place at accelerated stability testing conditions of 40 °C ± 2 °C and 75 ± 5 % relative humidity (RH). To assay the formulation and determine content uniformity over the three-month storage period, a suitable stability-indicating reversed-phase high performance liquid chromatography (RP-HPLC) method was developed and validated. Other cream properties which were tested included: qualitative aspects, viscosity, pH and microbial limits. At the end of the three-month stability testing period, the cream was found to meet most of the specifications set, except for cream homogeneity and viscosity. A TBH cream formulation was thus developed and optimised to meet a certain viscosity specification. Although this formulation was found to meet the viscosity specification on the day after its manufacture, the viscosity was found to increase on storage, such that it was outside the set viscosity specification range.
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