This effect was attributed to the presence of at least one p-methoxybenzyl substituent in the structure. I would like to thank Dr. Catherine Kaschula, Professor Alessandra Pana, Ph.D. Molahlehi Sonopo, Ph.D. Jan Rijn Zeervaart and the IDMM laboratory for their contributions to the project.
Review of Biological and Synthetic aspects of Ajoene
- Introduction
- Overview of Garlic
- Organosulfur Compounds Found in Garlic
- Ajoene
- Cancer
- Treatment of cancer
- The Eukaryotic cell-cycle
- Apoptosis
- Other Causes of Apoptosis
- Anti-cancer Activity of Ajoene
- Anti-mutagenic
- Anti-tumour
- Chemosensitization
- Signaling pathways
- Mode of Action of Ajoene
- Modification of Microtubule Formation and Cell-Cycle Arrest
- Modification of Enzymes
- Summary
These interactions lead to the activation of the caspase cascade, which ultimately leads to cell death by degrading proteins necessary for cell viability. Ajoene possesses a variety of therapeutic benefits, its anti-cancer properties were of most relevance to the present study and some of the benefits have been detailed.
Synthesis aspects of Ajoene
- Overview
- Block’s Biomimet ic Synthesis
- Terminally-substituted Ajoene analogues
- University of Cape Town (UCT) synthesis
- Step 1 - Propargylation
- Step 2 - Radical Addition
- Sulfenylating Agent
- S -Sulfenylation Step
- Step 4 - Oxidation
- Summary
- Ajoene Analogues with Water-solubility-promoting Substituents
- Overview
For consistency in this thesis, the ajoene analogues are drawn with the sulfoxide on the left side of the molecule and the disulfide on the right as depicted in Figure 11. Thus, the "left side of the molecule" indicates the R1 group on the sulfoxide side , while the "right side of the molecule". In view of the sharp nature of thiols, it was more convenient to propargylate in situ.
The second step involves the regioselective radical addition of thiolacetic acid to the terminal of the alkyne of 1 to form a vinyl thioacetate using ACCN (1,1'-azobis(cyclohexane-1-carbonitrile)) as a radical initiator. The right side of the molecule is made up of an S-tosyl sulphenylating agent which itself is derived from a suitable R2 halide. Interestingly, having a p-fluorobenzyl substituent yielded a significantly less active analog, suggesting that having electron-withdrawing groups at the terminal reduces the activity of the central pharmacophore.
At the end of the study it was found that there was no significant effect on tumor growth in the subjects who received drug treatment compared to the control subjects and also that the drug had no negative effects on the health of the subjects. The synthesis began with attempts to synthesize 6' ajoenes with a free phenol on the left side of the molecule.
Results and Discussions
Studies towards synthesis of Ajoene 6’
At this point it was decided to try to exploit the SN1 character of the benzyl position for direct substitution with a sulfur nucleophile. Reaction was allowed to proceed for 30 min to facilitate formation of the p-quinone methide before thioacetic acid was added dropwise to the mixture. The reaction was monitored by TLC, following the disappearance of the polar benzyl alcohol, which indicated that it had reached completion within the first hour.
39 deprotonation of the acidic phenolic hydroxyl group to the phenoxide ion, which retarded the ready hydrolysis of the thioacetate via electronic repulsion with the hydroxide ion. From the findings described, it became clear that using the p-hydroxybenzyl starting material to access vinyl thioacetate 8 was problematic due to the electronic connection between the p-hydroxyl group and the benzyl position. The reaction involves the formation of an alkoxyphosphonium ion from the more reactive hydroxyl group, followed by iodide ion substitution with expulsion of triphenylphosphine oxide.
The reaction was driven by the formation of triphenylphosphine oxide and was complete in 1.5 h as seen on TLC with the disappearance of the alcohol and the formation of the less polar iodide along with the oxide. Again, the difficulty in the sequence may have arisen from the formation of the phenoxide ion in the presence of KOH, resulting in the deactivation of subsequent chemistry.
Synthesis of Ajoenes, 6
- Thiotosylate Sulfenylating Agent, 19
- PMB Vinyl thioacetate, 23 35
- PMB-phenol Disulfide, 25
- PMB-phenol Sulfoxide, 26
The next step involved converting the primary hydroxyl group to the corresponding iodide. Analysis of the 13C NMR spectrum of 22 revealed that the alkyne carbons resonate at 80.1 ppm and 71.3 ppm, corresponding to C-7 and C-8, respectively. After one hour, TLC analysis showed the formation of a more polar site and significant depletion of the starting alkyne 22 .
However, two vinyl doublets of triplets (allylic coupling to H-5) were observed downfield at 6.67 ppm and 6.50 ppm corresponding to H-3 of the E and Z isomers, respectively (see Figure 18 for numbering), proving that that addition had taken place at the terminus of alkyne 22. The 13C NMR spectrum showed all the 26 necessary carbon signals (13 for each isomer), whereby two downfield signals were observed at 193.1 ppm and 191.4 ppm corresponding to thioacetate carbonyl (C -2) in the E - respectively Z isomers. The coupling (see Scheme 22) of vinyl thioacetate 23 with the sulfenylating agent 19 to give the core vinyl disulfide pharmacophore was a critical step in the synthesis of the target molecule 6.
The final step in the synthesis of the target sulfoxide 26 (see Scheme 24), involved the chemoselective oxidation of the sulfide precursor 25. The 13C NMR spectrum further supported the oxidation reaction, whereby there was a field shift from the methylene carbons α- to the sulfoxide.
Increasing the Aqueous Solubility of Substituted Ajoenes
- Introduction
- PMB-Acetate, 27
- PMB-Ester, 28
- PMB-Amide, 29
- Overview and Comments
The 1H NMR spectrum for PMB-acetate 27 (see Figure 24) confirmed that the desired substitution was achieved due to the observed signals of phenol 26, together with a new singlet at 2.28 ppm for both Z/E-isomers corresponding to the acetyl methyl group of acetate. Furthermore, the presence of carbonyl was confirmed by carbonyl stretching in the IR spectrum at 1700 cm-1. The second target was accessed by O-alkylation of sulfoxide 26 via an SN2 reaction (see Scheme 26).
The chemoselective manner exhibited in the substitution reaction, i.e. attacking the methylene rather than the ester carbonyl, was due to the greater electrophilicity of the methylene carbon due to the presence of two electron withdrawing groups. All other core structure signals were also observed and used in distinguishing the structure of the product. In addition, the presence of the carbonyl was confirmed by a clear carbonyl stretch in the IR spectrum at 1700 cm -1 .
It was postulated that this may have been due to one of the relatively acidic protons α- to the sulfoxide undergoing deprotonation by NaH to form a delocalized allylic anion. The reduced reactivity in the reaction compared to that of 28 can be attributed to the nature of the electrophile, as the amide group is not as electron-withdrawing as its ester counterpart due to back-donation of nitrogen.
Biological and Solubility Evaluation of the Substituted Ajoenes
- Determination of the IC 50 of Ajoene analogues
- Assessment of Aqueous Solubility by Measuring Turbidity
- Animal study ( in vivo )
- Ajoene analogues as Chemosensitizing agents
- Overview and Comments
The next section discusses the solubility studies and the animal study performed on one of the analogs. The intensity of the scattered light is dependent on the concentration of scattering particles, and the turbidity is measured using a UV-vis spectrophotometer at 620 nm. 66 Indeed, turbidimetric analysis of the new analogs revealed an increase in water solubility compared to bis-PMB, which became cloudy at a concentration of about 20 M.
67 Figure 29: A constant increase in the weight of the mice from the treatment and control groups was observed during the study, indicating that the drug was not cytotoxic to the mice. The poor bioavailability due to solubility was thus ruled out as an explanation for the failure. The two studies involving both bis-PMB and the PMB-amide have shown that administration of the drug via intraperitoneal injection is not the best method and as such. oral or intravenous administration should be considered. For the former, the CC50 of Vincristine (the concentration required to reduce viability by 50% of KBV20C cells) was 0.85 M, which decreased eighteen-fold to 0.047 M when 6.67 M of the PMB allyl was combined with VCR .
Importantly, 6.67 M was below the CC50 of PMB-allyl 32 of 8.5 M, meaning that the reduced CC50 of the drug in combination was not due to the ajoene derivative. These results show how the addition of ajoene analogues activates the sensitivity of the tumor cells to the drug, resulting in a reduction in the concentration of VCR required to achieve 50% inhibition.
Summary and Future work
Experimental Section
General synthetic methods
The reaction was allowed to proceed for 2 hours, after which a more polar spot was observed on TLC. The reaction was allowed to proceed for 2 hours, after which a point-to-point conversion was observed on TLC (EtOAc:hexane, with the resulting product being more polar than the starting 4-(-2-iodoethyl)phenol. The reaction was carefully monitored by TLC (EtOAc:hexane = 10:90), which revealed the formation of a more polar site corresponding to thioacetate after one hour.
The reaction was quenched with saturated ammonium chloride (5 mL), the solvents were removed under vacuum on a rotary evaporator, and the resulting residue was extracted into DCM (15 mL x 3). The reaction was allowed to proceed for 2 h under -60 °C, producing a more polar product spot after TLC monitoring (EtOAc:Hexane = 80:20). Saturated Na2CO3 (5 mL) was added at -60 °C and the reaction was allowed to warm to room temperature.
DCM (25 mL) was added and washed with 1M HCl (2 mL) to remove NEt3 and DMAP, followed by the NaHCO3 (5 mL) to neutralize the reaction mixture. The reaction was heated at 45°C and closely monitored by TLC (EtOAc) which revealed the formation of a more polar spot corresponding to the amide product after 12 h.
Biological Experimental
- General
- Cell proliferation analysis
- Animal Study
After plating, the cells were allowed to settle and grow overnight in an incubator at 37°C. Each dilution was further diluted 100-fold in media, after which 10 liters of the newly formed dilution was added to the cells (see Table 6), giving a final drug concentration of 1 in 1000 of the original solutions and a DMSO of 0.1 %. On day 4, 10 L of MTT reagent was added to each well and the cells were incubated for an additional 4 hours at 37°C, followed by the addition of Solubilization Reagent (10% SLS at 0.01 M HCl) (100 µl) and at least finally incubated overnight at 37°C.
Background absorbance (with medium only) was then subtracted from each reading and the data analyzed using Graphpad Prism 4 using non-linear regression analysis fitted to a sigmoidal dose-response curve with A595 nm - baseline versus Log C to give IC50 at 95% confidence interval. 100 µl of the suspension (2.50 x 10 6 ) was then injected into the right hindquarter of each nude mouse. 88 drug doses were prepared by dissolving the test drug in 2.5% chondroitin, 10% DMSO, and PEG400 to make a solubilized solution and then administered to mice intraperitoneally from days 1 to 21.
The treated mice were monitored for signs of discomfort after the administration of the test drug, and as such their health, weight and tumor size (measured with calipers) were carefully recorded. After completion of the study on day 21, the mice were euthanized with halothane and weighed, and tumors were carefully removed and their volumes determined by (length 2 x height)/2.53.
