1. Synthesis of a Sulfur Variant for Treatment of Trypanosomiasis
Synthesis of a Sulfur Variant for Treatment of Trypanosomiasis
Carlos Vera-Esquivel; Dr. James Hagen
University of Nebraska at Omaha, Department of Chemistry
I. Introduction
Human African Trypanosomiasis (HAT) is a parasitic protozoan infection (Trypanosoma genus) common in 36 sub-Saharan African countries.
20,000 cases reported last year for HAT infection and another 65 million currently at risk.
The goal of this project is to synthesize an analog of the lead compound effective against Trypanosomiasis brucei rhodisiense (T.b. rhod) with low human toxicity.
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II. Available Treatments
Pentamidine – targets 1st stage T.b. gambiense
Hypo/Hyperglycemia
Hypotension
Leukopenia
Melarsoprol – effective against 2nd stage T.b. gambiense and 2nd stage T.b. rhod
Toxic
Brain Tumor Cysts
Encephaloapathy
Suramin - only available treatment against 1st stage T.b. rhodisiense.
Anaphylactic Shock
Renal Failure
Nifurtimox /Eflornithine - only for 2nd stage T. Gambiense.
G.I. Problems
Bone Marrow Toxicity
III. Chemical Results
Using 1.1 eq. morpholine and 1.4 eq. acetoxy borohydride CVE 51-6 was converted to CVE Three sequential recrystallizations with EtOAc, MeOH, and toluene gave the product.
Reagents were heated for 60 min. TLC indicated plenty of unreacted material remaining. Another 40 min heating was applied at 68.4 °C which improved TLC and thus yield.
After a 3 h reaction, a solid was precipitated by lowering the pH = 2 on ice.
The target bis-salt proved difficult to recrystallize from EtOAc, toluene, MeOH, and acetonitrile; therefore, chromatography was used.
Salt bath was applied for 4 h. Results matched literature.
Heating at 120 °C and 19 h gave carboxylic acid and alcohol formed by Cannizzaro reaction. Temperature and time were reduced to 115 °C for 10 h which stopped the Cannizzaro.
IV. Methodology
Reaction Tracking
Reactions were tracked using 1H-NMR, Mass Spectrometry, and TLC.
Structure Confirmation
Utilized GC-Mass Spec, 1H-NMR, 13C-NMR
Combustion analysis was performed by H-M-W Laboratories
Purification
Impurities were removed by simply recrystallizing.
Biological Testing
Swiss Tropical and Public Health Institute.
IC50 is amount of compound that kills half of the trypanosomes.
SI (selectivity index) = IC50 toxicity/IC50 activity
The oxygen variant (J33-2) showed a 5 fold greater selectivity for T.b rhod. when compared to the sulfur analog (CVE ).
CVE appears to be 590 fold less toxic than Melarsoprol; much improvement.
The oxygen to sulfur change of CVE seems to increase killing efficacy of trypanosomes at the cost of increasing toxicity towards human cells. (Table 1).
VII. References
Trypanosoma (2010, May 27) The Australian Society for Parasitology.
HAT factsheet,WHO, Jan WHO website accessed February 2017.
Jamonneau, V.; Ilboudo, H.; Kabore, J.; Kaba, D.; Koffi, M.; Solano, P.; Garcia, A.; Courtin, D.; Laveissiere, C.; Lingue, K.; Buscher, P.; Bucheton, B. Untreated Human Infections by Trypanosoma brucei gambiense Are Not 100% Fatal. Plos Neglect. Trop. Dis. 2012, 6, e1691.
Abdel-Magid, A. F.; Carons, K. G.; Harr, B.D.; Maryanoff, C. A.; Shah, R. D.; Reductive Amination of Aldehydes and Ketones with Sodium Triacetoxyborohydride. Studies on Direct and Indirect Reductive Amination Procedures The Journal of Organic Chemistry 1996, 61 (11), 3849–3862.
Sykes, M. L., Baell, J. B., Kaiser, M., Chatelain, E., Moawad, S. R., Ganame, D., Ioset, J. R., … Avery, V. M. (2012). Identification of compounds with anti-proliferative activity against Trypanosoma brucei brucei strain 427 by a whole cell viability based HTS campaign. PLoS neglected tropical diseases, 6(11), e1896.
V. Biological Results
VI. Conclusion ID
Activity IC50 µM (T. b. rhod) (SI vs L6)
Cytotoxicity L6 IC50 (µM)
HFF IC50, 𝛍M (SI)
U-2 OS IC50, 𝛍M (SI)
HC-04 IC50, 𝛍M (SI)
J33-2
0.53 (150) 81
400 (750)
320 (600)
300 (570)
CVE
5.32
17.16 -
CVE
0.33 (30) 10
184.8 (560)
149.0 (450)
128.0 (390)
Melarsoprol
0.006 (2.9)
0.017
Table 1 – Toxicity and selectivity of compounds: Toxicity was determined for Rat Skeletal Myoblast Cells (L6). SI were determined with relative IC50 values of T.b. rhod., Human Foreskin Fibroblasts (HFF), Human Bone Osteosarcoma Epithelial Cells (U-2 OS), and Human Hepatocytes (HC-04) to L6 cells.
Acknowledgements
Special thank you to the chemistry department for purchasing starting materials and quantitative analysis services.