Cytotoxic Alkaloids from Microcos paniculata with Activity at Neuronal Nicotinic
Receptors
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy of the Graduate School of The Ohio State University
By
Patrick Colby Still
Graduate Program in Pharmacy
The Ohio State University
2013
Dissertation Committee:
A. Douglas Kinghorn, Advisor
Esperanza J. Carcache de Blanco
Karl Werbovetz
Edward J. Behrman
Copyright by
Patrick Colby Still
2013
Abstract
Natural products have a long history of use as leads for drug discovery. The
World Health Organization (WHO) estimates that in Africa for example, up to 80% of the population relies on plant-derived traditional medicines to help meet primary healthcare needs (WHO, 2002). In the area of cancer, of the 175 new chemical entities in the western drug market from the 1940s to 2010, 48.6% were a combination of unaltered natural products, semisynthetic natural product derivatives, or obtained by total synthesis inspired by a natural product skeleton (Newman and Cragg, 2012). The question of why secondary metabolites are biosynthesized by organisms is still a topic of debate.
However, ecological research has suggested that these compounds play a role as defense agents and are used also in communication (Williams et al., 1989; Yi et al., 2003; Chen et al., 2009).
This dissertation work emcompasses bioactive alkaloids from the plant Microcos paniculata collected in Vietnam. Microcos paniculata is a large shrub or small tree that grows in several countries in South and Southeast Asia. Three new piperidine alkaloids, microgrewiapines A-C, and a number of known compounds were isolated from cytotoxic fractions of the separate chloroform-soluble extracts of the stem bark, branches, and leaves of M. paniculata. Comprehensive structure elucidation techniques were used to elucidate and identify the structure of the new compounds. In addition, 1H -13C H2BC ii
NMR spectroscopy and induced circular dichroism (ICD) were employed to confirm the structures of the known compounds maslinic acid and (-)-loliolide. A total synthesis of
2 -methyl-3,6 -(di-tert-butyldimethylsilyl) piperidine was accomplished to investigate the structural requirements for biological activity. The isolated compounds exhibited a range of cytotoxic potencies against the HT-29 human colon cancer cell line that was used to screen extracts, chromatographically purified fractions, and pure compounds.
Structural similarities between the isolated piperidine alkaloids and certain previously identified nicotinic receptor (nAChR) antagonists, along with growing evidence to support the involvement of nAChRs in various aspects of cancer, provided a rationale to examine the effects of the isolates on nAChRs (Egleton et al., 2008; Schuller, 2009;
González-Cestari et al., 2009; Henderson et al., 2010). When evaluated for their effects on human 3 4 or 4 2 nAChRs, several of these compounds were shown to have antagonistic activity for both receptor subtypes.
As a result of this dissertation work, Microcos paniculata was collected as a tropical plant in Vietnam in 2008, leading to pure compounds being characterized showing activity at the cellular and receptor levels. The cytotoxicity and nAChR modulatory activity of compounds isolated in this investigation represent the first time such bioactivies have been shown for the constituents of a plant in the genus Microcos.
Contemporary structure elucidation techniques were used and a preliminary analysis of the structural features contributing to the bioactivity of the isolated compounds has been made.
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This dissertation is dedicated to my Dad, who from a young age instilled a desire in me to
ask questions, and to look where I’m going, and not where I’ve been.
iv
Acknowledgments
I would like to thank, first and foremost, my thesis dissertation advisor Dr. A.
Douglas Kinghorn. Throughout my graduate work, he has cultivated in me a sense of scientific excellence and patience. His style and way of thinking represent a model for future challenges I will face as a scientist. I also wish to thank Dr. Li Pan. It was a great privilege to witness her keen ability to envision a natural product structure from the data presented in NMR spectra, and she is thanked for her patience in instilling that ability within me. In addition, Dr. Esperanza J. Carcache de Blanco is thanked for her guidance and valued conversations about science and life, and for information on the mitochondrial membrane potential assay. I also wish to thank Dr. James R. Fuchs for his guidance with the synthetic work contained in this thesis. His energy, encouragement and outstanding teaching ability were inspirational. Dr. Karl Werbovetz and Dr. Edward Behrman are also thanked for taking the time to review this work, and for being a part of this dissertation committee.
Microcos paniculata samples were collected in Vietnam under the terms of an agreement between the University of Illinois at Chicago (UIC) and the Institute of
Ecology and Biological Resources (IEBR) of the Vietnam Academy of Science and
Technology, Hanoi, Vietnam. I wish to thank Dr. Djaja Djendoel Soejarto (UIC) for
v providing three parts of M. paniculata for this dissertation work, and for his enthusiastic and knowledgeable discussions about plant taxonomy. I am grateful to Mr. John Fowble and Dr. Craig McElroy, College of Pharmacy, The Ohio State University (OSU), and Dr.
Chunhua Yuan, for many discussions and practical guidance on NMR spectroscopy, as well as Ms. Nan Kleinholz, Mr. Mark Apsega, and Dr. Kari Green-Church, Mass
Spectrometry and Proteomics Facility, OSU for assisting with LC-MS dereplication and mass spectrometry.
I am grateful to Dr. Heebyung Chai for HT-29 human colon cancer cell cytotoxicity testing, and the mutual satisfaction we shared after determining the main active compound of M. paniculata using bioactivity-guided fractionation. I also wish to thank Dr. Dennis McKay, Dr. Tatiana F. González-Cestari, and Ms. Bitna Yi, Division of
Pharmacology, College of Pharmacy, OSU for their guidance in the use of pharmacological assays on nicotinic receptor subtypes and for their collaboration.
Working in their laboratory broadened my scope of knowledge and took my isolated compounds to new heights. Emeritus Professor Dr. Popat Patil, Division of
Pharmacology, College of Pharmacy, OSU is thanked for his inspiration and guidance throughout the pharmacological investigation.
Sources of financial support for this dissertation work are gratefully acknowledged. These sources of support include the Raymond Doskotch Fellowship in
Natural Products Chemistry (Division of Medicinal Chemistry and Pharmacognosy,
College of Pharmacy, OSU; 2010-2011), grant P01CA125066 awarded to Dr. A. Douglas
Kinghorn, from the National Cancer Institute, National Institutes of Health (NCI, NIH),
vi
Bethesda, Maryland (2007-2012), and an American Society of Pharmacognosy 2012
Student Travel Grant.
I am privileged to have worked with many colleagues throughout this dissertation work who provided support each step of the way. In particular, I wish to thank Dr. Mark
Bahar, Ms. Annecie Benetrehina, Ms. Lynette Bueno, Dr. Joshua Fletcher, Dr. Brandon
Henderson, Dr. Nivedita Jena, Dr. Chenglong Li, Mr. Jie Li, Mr. Ben Naman, Mr. Pretiq
Patel, Mr. Ryan Pavlovicz, Dr. Yulin Ren, and Mr. John Woodard.
I also wish to acknowledge my family and closest friends for the encouragement and pride they have shown me. This dissertation work honors the scientific accomplishments of two family members, both with their own achievements in the field of Pharmacognosy. James Still (1812-1885), one of my ancestors and a self-taught healer who travelled the New Jersey pinelands, and saw patients from across the Philadelphia region, practiced herbal medicine in the formative years of the United States of America.
My cousin, the late Dr. Cecil C. Still, Department of Biochemistry and Microbiology,
Cook College, Rutgers University, catalogued the medicinal plants of New Jersey and the
Philadelphia region in his book, Botany and Healing. I cherish the personally signed copy of his book, and remember his pride in me for continuing in the family tradition of
Pharmacognosy. I thank my grandmother, Mrs. Delores Hawkins, for teaching me about gardening, which formed the basis of my appreciation for plant natural products. My good friends, Mr. Ryan P. Mouton and Mr. Julian Richard, are sincerely thanked for their endless encouragement, and helping me to recognize the best parts of myself.
vii
Vita
EDUCATION
2003...... Colonial Forge High School, Stafford,
Virginia
2007...... B.S. Chemistry, Cum Laude, Virginia
Commonwealth University, Richmond, Virginia
2007-2013 ...... Ph.D. Student, College of Pharmacy, The
Ohio State University, Columbus, Ohio
HONORS AND AWARDS
1. David F. Ingraham Fellowship in Chemistry, Department of Chemistry, Virginia
Commonwealth University, Richmond, Virginia, 2006.
2. Raymond W. Doskotch Fellowship in Natural Products Chemistry, College of
Pharmacy, The Ohio State University, Columbus, Ohio, 2010.
3. American Chemical Society Certificate of Innovation, Graduate Student Summer
Institute on Technology Development, Washington, DC, 2010.
4. The Mary Frances Picciano, Dietary Supplement Research Practicum, National
Institutes of Health, Bethesda, Maryland, 2012.
viii
5. American Society of Pharmacognosy, Student Travel Award, International Congress
on Natural Products Research, New York, New York, 2012
NATIONAL MEETING PRESENTATIONS
1. Chiu, W. L., Peters, G. A., Levielle, G., Still, P. C., Cousins, S., Osborne, B., Elhai, J.
“The Role of Flavonoids in the Gunnera-Nostoc Symbiosis. University of Maryland
Molecular Biology Symposium, Arabidopsis thaliana Research Initiative at
University of Maryland, ATRIUM; College Park, Maryland, June 2004; poster
presentation.
2. Still, P. C., Pan, L., Ninh, T. N., Soejarto, D. D., Chai, H.-B., Fuchs, J. R., Carcache
de Blanco, E. J., Kinghorn, A. D. “Bioactivity-guided Isolation of Microgrewiapine, a
Cytotoxic Piperidine Alkaloid from Grewia paniculata”. 52nd Annual Meeting of the
American Society of Pharmacognosy; San Diego, California, August 2011; poster
presentation.
3. Still, P. C., Yi, B., Pavlovicz, R., González-Cestari, T. F., Pan, L., Chai, H.-B., Ninh,
T. N., Soejarto, D. D., Li, C., Fuchs, J. R., McKay, D., Kinghorn, A. D. “Alkaloids
from Microcos paniculata with Cytoxtoxic and Non-competitive Nicotinic Receptor
Antagonistic Activities”. International Congress on Natural Products Research, New
York, New York, August 2012; oral presentation.
PUBLICATIONS
1. Chiu, W.-L., Peters, G. A., Levieille, G., Still, P. C., Cousins, S., Osborne, B., Elhai,
J. Nitrogen Deprivation Stimulates Symbiotic Gland Development in Gunnera
manicata. Plant Physiology. 2003, 139, 224-230.
ix
2. Lucas, D. M., Still, P. C., Bueno-Pérez, L., Grever, M. R., Kinghorn, A. D. Potential
of Plant-Derived Natural Products in the Treatment of Leukemia and Lymphoma.
Current Drug Targets. 2010, 11, 812-822. (review article)
3. Ren, Y., Wei, M., Still, P. C., Yuan, S., Deng, Y., Chen, X., Himmeldirk, K.,
Kinghorn, A. D. Synthesis and Antitumor Activity of Ellagic Acid Peracetate. ACS
Medicinal Chemistry Letters. 2012, 3, 631-636.
4. Still, P. C., Yi, B., Pavlovicz, R., González-Cestari, T. F., Pan, L., Chai, H.-B.,
Ninh, T. N., Soejarto, D. D., Li, C., Fuchs, J. R., McKay, D., Kinghorn, A. D.
Alkaloids from Microcos paniculata with Cytoxtoxic and Nicotinic Receptor
Antagonistic Activities. Journal of Natural Products. 2013, 76, 243-249.
Fields of Study
Major Field: Pharmacy
x
Table of Contents
Cytotoxic Alkaloids from Microcos paniculata with Activity at Neuronal Nicotinic Receptors...... 1
DISSERTATION ...... 1
Abstract ...... ii
Acknowledgments...... v
Vita ...... viii
Fields of Study ...... x
Table of Contents ...... xi
List of Tables ...... xiv
List of Figures ...... xv
List of Schemes ...... xix
List of Abbreviations ...... xx
Chapter 1: Discovery of Cancer Chemotherapeutic and Nicotinic Receptor (nAChR)
Drugs of Natural Origin ...... 1
A. Plant-derived Anticancer Drugs ...... 1
1. Vinca (Catharanthus) alkaloids ...... 3 2. Epipodophyllotoxin derivatives ...... 4 3. Taxanes ...... 5 4. Camptothecins...... 6 xi
5. Omecetaxine mepesuccinate (homoharringtonine) ...... 7 6. Trastuzumab emtansine ...... 8 7. Ingenol mebutate ...... 9 B. Nicotinic Acetylcholine Receptor (nAChR) Agents from Natural Sources ...... 13
1. Drug discovery approches related to nAChR action ...... 18 2. Natural sources of nAChR agonists and antagonists ...... 19 Chapter 2: Taxonomy and Phytochemistry of Microcos paniculata ...... 28
A. Background on Microcos ...... 28
1. The family Malvaceae ...... 28 2. Nomenclature and classification for the genus Microcos and the closely related genus Grewia ...... 29 3. Genus Microcos and Microcos paniculata L...... 30 4. Overview of phytochemical studies on the genus Microcos ...... 33 Chapter 3: Phytochemical and Bioactivity Studies on the Stem Bark, Branches, and
Leaves of Microcos paniculata ...... 42
A. Statement of Problem ...... 42
B. Experimental ...... 42
1. General experimental procedures ...... 42 2. Plant material ...... 44 3. Extraction of the stem bark, branches, and leaves of Microcos paniculata 45 4. Chromatography of the chloroform-soluble extracts of the stem bark, branches and leaves of Microcos paniculata ...... 49 5. Dereplication of the stem bark, branches, and leaves of M. paniculata ..... 58 6. Characterization of isolated compounds ...... 64 7. Characterization of synthetic compounds ...... 72 8. Cytotoxicity and nAChR antagonistic activity of isolated alkaloids from M. paniculata and synthetic analogues ...... 77 C. Discussion ...... 81
xii
1. General points on the characterization of the compounds isolated from M. paniculata ...... 81 2. Structure elucidation of microgrewiapine A (56) ...... 82 3. Structure elucidation of microgrewiapine B (60) ...... 91 4. Structure elucidation of microgrewiapine C (61) ...... 97 5. Identification of microcosamine A (64) ...... 102 6. Identification of liriodenine (59) ...... 106 7. Identification of 7 -(3 ,4 -dihydroxyphenyl)-N-[4-methoxyphenyl)ethyl] propenamide (58)...... 111 8. Identification of maslinic acid (62) ...... 116 9. Identification of palmitic acid (57) ...... 124 10. Identification of (-)-epicatechin (34) ...... 126 11. Identification of daucosterol ( -sitosterol-3- -D-glycoside) (63) ...... 130 12. Identification of (-)-loliolide (65) ...... 133 13. Synthesis of 2 -methyl-3 ,6 -(di-tert-butyldimethylsilyl)piperidine: overview of synthetic route ...... 139 14. Identification of (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-hydroxy-1- heptene (66)...... 140 15. Identification of (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-(tert- butyldimethylsilyl)-1-heptene (67)...... 142 16. Identification of (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-(tert- butyldimethylsilyl)-1,2-diol (68)...... 143 17. Identification of (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-(tert- butyldimethylsilyl)-1-(tert- butyldimethylsilyl)-2-ol (69)...... 145 18. Identification of (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-(tert- butyldimethylsilyl)-1-(tert-butyldimethylsilyl)-2-heptanone (70)...... 146 19. Identification of of (2R,3R,6R)-3-[(tert)-butyldimethylsilyl)]-2-methyl-6- (tert-butyldimethylsilyl)piperidine (72)...... 149 20. Biological test data obtained ...... 154 D. Conclusions ...... 159
References ...... 162
Appendix A: Cell Culture and Cytotoxicity Assays ...... 174
Appendix B: Calcium Accumulation Assays ...... 177 xiii
List of Tables
Table 1.1. Plant-derived anticancer compounds currently on the market ...... 12
Table 1.2. Natural products with activity at nicotinic acetylcholine receptors (nAChRs)
...... 27
Table 2.1. Flavonoids and other phenolics isolated from Microcos paniculata ...... 34
Table 2.2. Piperidine alkaloids isolated from the genus Microcos and representative piperidines from other plant and animal taxa ...... 37
Table 2.3. Triterpenoids isolated from the genus Microcos ...... 39
Table 3.1. Dereplication of M. paniculata chloroform extract of the leaves ...... 61
Table 3.2. Dereplication of M. paniculata chloroform extract of the stem bark ...... 62
Table 3.3. Dereplication of M. paniculata chloroform extract of the branches ...... 63
Table 3.4. Cytotoxicity assay results of M. paniculata extracts, fractions, and pure compounds against HT-29 human colon cancer cells ...... 79
Table 3.5. Percent inhibition of nAChR activity by test compounds applied at a 10 µM dose ...... 80
Table 3.6. Effects of isolated and synthesized antagonists on human nAChRs ...... 80
xiv
List of Figures
Figure 1.1. Structures of plant-derived anticancer drugs currently on the market and selected related compounds ...... 10 Figure 1.2. Topology of the nAChR subunits…………………………………………... 15 Figure 1.3. nAChR resides lining the ion pore and charge selectivity ...... 17 Figure 1.4. Structures of compounds with activity at nAChRs ...... 25 Figure 2.1. Illustration of Microcos paniculata L...... 32 Figure 2.2. Photograph of Microcos paniculata collected from Kego Nature Reserve, Hatinh Province, Vietnam, in December 2008 ...... 33 Figure 2.3. Structures of flavonoids and other phenolics isolated from Microcos paniculata ...... 35 Figure 2.4. Structures of piperidine alkaloids isolated from Microcos spp. and representative piperidines from other plant and animal taxa ...... 38 Figure 2.5. Structures of triterpenoids isolated from genus Microcos ...... 40 Figure 3.1. Fractionation tree employed for Microcos paniculata stem bark ...... 46 Figure 3.2. Fractionation tree employed for Microcos paniculata branches ...... 48 Figure 3.3. Fractionation tree employed for Microcos paniculata leaves ...... 49 Figure 3.4. Summary of compounds isolated from M. paniculata stem bark, branches, and leaves in the present investigation...... 57 Figure 3.5. Numbering system used for piperidine alkaloids isolated from M. paniculata ...... 81 Figure 3.6. Chemical shift difference values (S-R), for protons on the esterified piperidinol ring of 56, as established by a modified Mosher ester method ...... 85 1 Figure 3.7. H NMR spectrum of microgrewiapine A (56) (CDCl3, 600 MHz) ...... 85 1 Figure 3.8. H NMR spectrum of microgrewiapine A (56) (CDCl3, 600 MHz; expanded scale) ...... 86 13 Figure 3.9. C NMR and spectrum of microgrewiapine A (56) (CDCl3, 150 MHz) ...... 86 13 Figure 3.10. C NMR spectrum of microgrewiapine A (56) (CDCl3, 150 MHz; expanded scale) ...... 87 Figure 3.11. DEPT spectrum of microgrewiapine A (56) (CDCl3) ...... 87 Figure 3.12. DEPT spectrum of microgrewiapine A (56) (CDCl3; expanded scale) ...... 88 Figure 3.13. HSQC spectrum of microgrewiapine A (56) (CDCl3) ...... 88 Figure 3.14. HSQC spectrum of microgrewiapine A (56) (CDCl3; expanded scale) ...... 89 Figure 3.15. HMBC spectrum of microgrewiapine A (56) (CDCl3) ...... 89 Figure 3.16. COSY spectrum of microgrewiapine A (56) (CDCl3) ...... 90 Figure 3.17. NOESY spectrum of microgrewiapine A (56) (CDCl3) ...... 90 Figure 3.18. Selected NOESY correlations of microgrewiapine B (60) (left) and energy- xv minimized structure (right) ...... 92 1 Figure 3.19. H NMR spectrum of microgrewiapine B (60) (CDCl3, 400 MHz) ...... 93 13 Figure 3.20. C NMR spectrum of microgrewiapine B (60) (CDCl3, 100 MHz) ...... 93 Figure 3.21. DEPT spectrum of microgrewiapine B (60) (CDCl3)...... 94 Figure 3.22. HSQC spectrum of microgrewiapine B (60) (CDCl3) ...... 94 Figure 3.23. HSQC spectrum of microgrewiapine B (60) (CDCl3; expanded scale) ...... 95 Figure 3.24. HMBC spectrum of microgrewiapine B (60) (CDCl3) ...... 95 Figure 3.25. COSY spectrum of microgrewiapine B (60) (CDCl3) ...... 96 Figure 3.26. NOESY spectrum of microgrewiapine B (60) (CDCl3) ...... 96 1 Figure 3.27. H NMR spectrum of microgrewiapine C (61) (CDCl3, 400 MHz) ...... 98 13 Figure 3.28. C NMR spectrum of microgrewiapine C (61) (CDCl3, 100 MHz) ...... 98 Figure 3.29. DEPT spectrum of microgrewiapine C (61) (CDCl3)...... 99 Figure 3.30. HSQC spectrum of microgrewiapine C (61) (CDCl3) ...... 99 Figure 3.31. HSQC spectrum of microgrewiapine C (61) (CDCl3, expanded sclae)...... 100 Figure 3.32. HMBC spectrum of microgrewiapine C (61) (CDCl3) ...... 100 Figure 3.33. COSY spectrum of microgrewiapine C (61) (CDCl3) ...... 101 Figure 3.34. NOESY spectrum microgrewiapine C (61) (CDCl3) ...... 101 1 Figure 3.35. H NMR spectrum of microcosamine A (64) (CDCl3, 600 MHz) ...... 104 Figure 3.36. DEPT spectrum of microcosamine A (64) (CDCl3) ...... 104 Figure 3.37. HSQC spectrum of microcosamine A (64) (CDCl3) ...... 105 Figure 3.38. HMBC spectrum of microcosamine A (64) (CDCl3) ...... 105 Figure 3.39. COSY spectrum of microcosamine A (64) (CDCl3) ...... 106 1 Figure 3.40. H NMR spectrum of liriodenine (59) (CDCl3, 400 MHz) ...... 108 13 Figure 3.41. C NMR spectrum of liriodenine (59) (CDCl3, 100 MHz) ...... 108 Figure 3.42. DEPT spectrum of liriodenine (59) (CDCl3) ...... 109 Figure 3.43. HSQC spectrum spectrum of liriodenine (59) (CDCl3) ...... 109 Figure 3.44. HMBC NMR spectrum of liriodenine (59) in CDCl3 ...... 110 Figure 3.45. COSY NMR spectrum of liriodenine (59) (CDCl3) ...... 110 Figure 3.46. 1H NMR spectrum of 7 -(3 ,4 -dihydroxyphenyl)-N-[4- methoxyphenyl)ethyl]propenamide (58) (MeOD, 400 MHz) ...... 112 Figure 3.47. 13C NMR spectrum (400 MHz) of 7 -(3 ,4 -dihydroxyphenyl)-N-[4- methoxyphenyl)ethyl]propenamide (58) (MeOD, 100 MHz) ...... 113 Figure 3.48. DEPT NMR spectrum of 7 -(3 ,4 -dihydroxyphenyl)-N-[4- methoxyphenyl)ethyl]propenamide (58) (MeOD) ...... 113 Figure 3.49. HSQC NMR spectrum of 7 -(3 ,4 -dihydroxyphenyl)-N-[4- methoxyphenyl)ethyl]propenamide (58) (MeOD) ...... 114 Figure 3.50. HMBC NMR spectrum of 7 -(3 ,4 -dihydroxyphenyl)-N-[4- methoxyphenyl)ethyl]propenamide (58) (MeOD) ...... 114 Figure 3.51. HMBC NMR spectrum of 7 -(3 ,4 -dihydroxyphenyl)-N-[4- methoxyphenyl)ethyl]propenamide (58) (MeOD, expanded scale) ...... 115 Figure 3.52. COSY NMR spectrum of 7 -(3 ,4 -dihydroxyphenyl)-N-[4- methoxyphenyl)ethyl]propenamide (58) (MeOD) ...... 115 1 Figure 3.53. H NMR spectrum of (-)-maslinic acid (62) (pyridine-d5, 400 MHz) ...... 117 1 Figure 3.54. H NMR spectrum of (-)-maslinic acid (62) (pyridine-d5, 400 MHz, xvi expanded scale) ...... 118 13 Figure 3.55. C NMR spectrum of (-)-maslinic acid (62) (pyridine-d5, 100 MHz) ...... 118 13 Figure 3.56. C NMR spectrum of (-)-maslinic acid (62) (pyridine-d5, 100 MHz, expanded scale) ...... 119 Figure 3.57. DEPT NMR spectrum of (-)-maslinic acid (62) (pyridine-d5) ...... 119 Figure 3.58. DEPT NMR spectrum of (-)-maslinic acid (62) (pyridine-d5, expanded scale) ...... 120 Figure 3.59. HSQC NMR spectrum of (-)-maslinic acid (62) (pyridine-d5) ...... 120 Figure 3.60. HSQC NMR spectrum of (-)-maslinic acid (62) (pyridine-d5, expanded scale) ...... 121 Figure 3.61. HMBC NMR spectrum of (-)-maslinic acid (62) (pyridine-d5) ...... 121 Figure 3.62. COSY NMR spectrum of (-)-maslinic acid (62) (pyridine-d5) ...... 122 Figure 3.63. NOESY NMR spectrum of (-)-maslinic acid (62) (pyridine-d5) ...... 122 Figure 3.64. ECD spectrum of (-)-maslinic acid (62) in a DMSO solution (red). ECD spectrum of compound 62 in a DMSO solution of Mo2(OAc)4 (blue)...... 123 4+ Figure 3.65. The O-C-C-O dihedral in the maslinic acid-[Mo2] complex ...... 123 1 Figure 3.66. H NMR spectrum of palmitic acid (57) (pyridine-d5, 400 MHz) ...... 125 13 Figure 3.67. C NMR spectrum of palmitic acid (57) (pyridine-d5, 100 MHz) ...... 125 13 Figure 3.68. C NMR spectrum of palmitic acid (57) (pyridine-d5, 100 MHz) (expanded scale) ...... 126 Figure 3.69. 1H NMR spectrum of epicatechin (34) (MeOD, 300 MHz) ...... 128 Figure 3.70. 13C NMR spectrum of epicatechin (34) (MeOD, 75 MHz) ...... 128 Figure 3.71. 13C NMR spectrum of epicatechin (34) (MeOD, 75 MHz, expanded scale) ...... 129 Figure 3.72. CD curve for (-)-epicatechin (34) ...... 129 1 Figure 3.73. H NMR spectrum of daucosterol (63) (pyridine-d5, 400 MHz) ...... 131 1 Figure 3.74. H NMR spectrum of daucosterol (63) (pyridine-d5, 400 MHz, expanded scale) ...... 131 13 Figure 3.75. C NMR spectrum of daucosterol (63) (pyridine-d5, 100 MHz) ...... 132 13 Figure 3.76. C NMR spectrum of daucosterol (63) (pyridine-d5, 100 MHz) (expanded scale, downfield region) ...... 132 13 Figure 3.77. C NMR spectrum of daucosterol (63) (pyridine-d5, 100 MHz) (expanded scale, upfield region) ...... 133 1 Figure 3.78. H NMR spectrum of (-)-loliolide (65) (CDCl3, 600 MHz) ...... 135 13 Figure 3.79. C NMR spectrum of (-)-loliolide (65) (CDCl3, 150 MHz) ...... 135 Figure 3.80. DEPT NMR spectrum of (-)-loliolide (65) (CDCl3) ...... 136 Figure 3.81. HSQC NMR spectrum of (-)-loliolide (65) (CDCl3) ...... 136 Figure 3.82. HMBC (CNST2, J filter = 10 Hz) NMR spectrum of (-)-loliolide (65) (CDCl3) ...... 137 Figure 3.83. HMBC (CNST2, J filter = 3 Hz) NMR spectrum of (-)-loliolide (65) (CDCl3) ...... 137 Figure 3.84. H2BC NMR spectrum of (-)-loliolide (65) (CDCl3) ...... 138 Figure 3.85. NOESY NMR spectrum of (-)-loliolide (65) (CDCl3) ...... 138 Figure 3.86. 1H NMR spectrum of (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-hydroxy- xvii
1-heptene (66) (CDCl3, 300 MHz)...... 141 Figure 3.87. 1H NMR spectrum of (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-(tert- butyldimethylsilyl)-1-heptene (67) (CDCl3, 300 MHz)...... 143 Figure 3.88. 1H NMR spectrum of (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-(tert- butyldimethylsilyl)-1,2-diol (68) (CDCl3, 300 MHz) ...... 144 Figure 3.89. NOESY NMR spectrum of (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5- (tert-butyldimethylsilyl)-1,2-diol (68) (CDCl3) ...... 145 Figure 3.90. 1H NMR spectrum of (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-(tert- butyldimethylsilyl)-1-(tert- butyldimethylsilyl)-2-ol (69) (CDCl3, 300 MHz) ...... 146 Figure 3.91. 1H NMR spectrum of (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-(tert- butyldimethylsilyl)-1-(tert-butyldimethylsilyl)-2-heptanone (70) (CDCl3, 300 MHz) ...... 148 Figure 3.92. COSY NMR spectrum (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-(tert- butyldimethylsilyl)-1-(tert- butyldimethylsilyl)-2-heptanone (70) (CDCl3) ...... 148 Figure 3.93. 1H NMR spectrum of (2R,3R,6R)-3-[(tert)-butyldimethylsilyl)]-2-methyl-6- (tert-butyldimethylsilyl)piperidine (72) (CDCl3, 300 MHz) ...... 150 Figure 3.94. 13C NMR spectrum of (2R,3R,6R)-3-[(tert)-butyldimethylsilyl)]-2-methyl-6- (tert-butyldimethylsilyl)piperidine (72) (CDCl3, 75 MHz) ...... 151 Figure 3.95. DEPT NMR spectrum of (2R,3R,6R)-3-[(tert)-butyldimethylsilyl)]-2- methyl-6-(tert-butyldimethylsilyl)piperidine (72) (CDCl3) ...... 151 Figure 3.96. HSQC NMR spectrum of (2R,3R,6R)-3-[(tert)-butyldimethylsilyl)]-2- methyl-6-(tert-butyldimethylsilyl)piperidine (72) (CDCl3) ...... 152 Figure 3.97. HMBC NMR spectrum of (2R,3R,6R)-3-[(tert)-butyldimethylsilyl)]-2- methyl-6-(tert-butyldimethylsilyl)piperidine (72) (CDCl3) ...... 152 Figure 3.98. COSY NMR spectrum of (2R,3R,6R)-3-[(tert)-butyldimethylsilyl)]-2- methyl-6-(tert-butyldimethylsilyl)piperidine (72) (CDCl3) ...... 153 Figure 3.99. NOESY NMR spectrum of (2R,3R,6R)-3-[(tert)-butyldimethylsilyl)]-2- methyl-6-(tert-butyldimethylsilyl)piperidine (72) (CDCl3) ...... 153 Figure 3.100. Effects of isolated and synthetic antagonists on recombinant nAChRs ...158 Figure 3.101. Concentration-response effects of isolated and synthetic antagonists on recombinant nAChRs ...... 158 Figure 3.102. Concentration-response effects of epibatidine in the absence or presence of compound microgrewiapine A (56) ...... 159
xviii
List of Schemes
Scheme 3.1. Synthesis of the 2 -methyl-3,6 -(di-tert-butyldimethylsilyl) piperidine analogue (72) ...... 140
xix
List of Abbreviations
1D, 2D: one- or two-dimensional
4 2: human neuronal nicotinic acetylcholine receptor subtype
3 4: human neuronal nicotinic acetylcholine receptor subtype
[ ]D: Specific optical rotation
Boc: tert-butyloxycarbonyl
CD: circular dichroism spectroscopy
COSY: correlation spectroscopy d: doublet dd: doublet of doublets ddd: doublet of doublet of doublets
(ppm): chemical shift in parts per million
C: carbon-13 chemical shift
H: proton chemical shift dt: doublet of triplets
DEPT: distortionless enchancement by polarization transfer
DMF: dimethylformamide
DMSO: dimethylsulfoxide
Glc: -D-glucopyranose
xx
H2BC: heteronuclear two-bond correlation spectroscopy
HEK293: human embryonic kidney 293 cells
HMBC: heteronuclear multiple bond correlation spectroscopy
HPLC: high-performance liquid chromatography
HRESIMS: high-resolution electrospray ionization mass spectroscopy
HSQC: heteronuclear single quantum coherence spectroscopy
Hz: hertz
IC50: sample concentration that inhibits cell growth by 50% compared to untreated control
IR: infrared spectroscopy
J: coupling constant
: wavelength in nanometer
M: molar concentration min: minute mp: melting point m/z: mass to charge ratio nAChR: nicotinic acetylcholine receptor
HBK: HEPES-buffered Krebs solution
NAM: negative allosteric modulator
NMO: N-methylmorpholine N-oxide
NMR: nuclear magnetic resonance nm: nanometer
NOESY: nuclear Overhauser enhancement spectroscopy xxi
ODS: octadecylsilane (C18) s: singlet
SRB: sulforhodamine B
SAR: structure-activity relationship
TBSCl: tert-butyldimethylsilyl chloride
Me3SiOTf: trimethylsilyl trifluoromethanesulfonate (TMS triflate) tR: retention time
UV: ultraviolet
(cm-1): infrared absorption frequency in reciprocal centimeters
WHO: World Health Organization
xxii
Chapter 1: Discovery of Cancer Chemotherapeutic and Nicotinic Receptor
(nAChR) Drugs of Natural Origin
A. Plant-derived Anticancer Drugs
Cancer is the leading cause of death in economically developed countries and is the second leading cause of death in developing countries (Jemal et al., 2011). According to 2008 GLOBOCAN statistics, which present cancer incidence for all cancers combined and for specific types of cancer for most countries of the world, 12.7 million new cancer cases and 7.6 million cancer deaths are estimated to have occurred in 2008 (Jemal et al.,
2011). Cancer incidence rates, when grouped by level of economic development, show that deaths due to lung, colon, prostate and breast cancer are most commonly seen in developed countries, while cancers of the lung, liver, cervix, uterus, and esophagus occur most frequently in developing countries (Jemal et al., 2011). The increase in worldwide deaths caused by cancer can be linked primarly to an aging population, and the adoption of cancer-causing behavior such as smoking (Jemal et al., 2011).
Anticancer drug discovery is not without a degree of paradox. The nitrogen mustard mechlorethamine, for example, was a chemical warfare agent initially intended for mass destruction, but is now in clinical use today. It was observed that in addition to the adverse skin reactions caused by nitrogen mustards, leukopenia was observed in humans, which prompted research into the potential of nitrogen mustards as antileukemic 1 drugs (Gilman, 1963). Mechlorethamine is currently in clinical use as a component of the mechlorethamine-vincristine-procarbazine-prednisone regimen for the treatment of
Hodgkin’s disease (Struck, 1995). Anticancer drug discovery involving natural product screening commenced in the 1950s through the Cancer Chemotherapy National Service
Center (CCNSC), established by the National Cancer Institute (NCI) (Mi et al., 2009).
Natural product extracts during this time were mainly screened against P388 and L1210 murine lymphocytic leukemia cells, followed by in vivo testing such as in a murine P388 lymphocytic leukemia model (Mi et al., 2009). A greater variety of tumor types for cytotoxicity screening was introduced with the NCI human cell line panel, composed of
60 different cancer cell types, including those of the prostate, lung, leukemia, melanoma, ovary, breast and kidney (Boyd and Paull, 1995; Mi et al., 2009). The 60 cell line panel tests compounds over a range of concentrations and accesses growth inhibition of the different cancer cell lines in the presence of the experimental compound. A “fingerprint” or profile of the cytotoxic activity of a pure compound or extract is obtained, and can be compared to the activity profile of known compounds, thereby providing some mechanistic information. A similar fingerprint of a new compound to a known compound may suggest common mechanisms of action or cellular targets (Mi et al., 2009).
The use of a cancer cell line to provide initial screens for cytotoxicity, or a
“gatekeeper,” in conjunction with high-performance liquid chromatography-mass spectrometry (LC-MS) may be used as a dereplication procedure to prioritize plant extracts for chromatographic purification, streamlining isolation efforts on extracts containing potentially new bioactive compounds. The sulforhodamine B assay (SRB) is
2 particularly amenable to inclusion in a LC-MS dereplication procedure. Plant extracts and pure compounds are stained with the dye sulforhodamine B, followed by liberation of this dye from the cells by washing with a tris base solution. An increase or decrease in the number of cells due to the effects of an applied test compound, results in a change in the amount of dye incorporated by the cells in the culture (Vichai and Kirtikara, 2006).
1. Vinca (Catharanthus) alkaloids
Just as with the nitrogen mustards, the anticancer activity of the vinca
(Catharanthus) alkaloids was also discovered by serendipity, when researchers at the
University of Western Ontario in Canada observed leukopenia and destruction of bone marrow in rats that were injected with a Catharanthus roseus (L.) G. Don (Apocynaceae) extract, as part of a search for new antidiabetic agents (Noble, 1961; Lucas et al. 2010).
The isolation and characterization of vinblastine (vincaleukoblastine) (1) by Noble, Beer, and Cutts, and vincristine (leurocristine) (2) by Svoboda and Neuss was accomplished in the late 1950s and early 1960s (Noble et al., 1958; Svoboda et al., 1959; Noble, 1961;
Neuss et al., 1962). The vinca (Catharanthus) alkaloids bind tubulin and block the process of mitosis, causing metaphase arrest (Johnson et al., 1960; Cutts, 1961). Early in vivo testing of 1 and 2 demonstrated size reduction of the tumors in mice engrafted with
L1210 and P1534 leukemia cells and Ehrlich ascites tumor cells, and served as evidence for antineoplastic activity (Cutts et al., 1960). Semi-synthetic derivatives of 1 and 2 such as vincristine sulfate (Oncovin®) and vinblastine sulfate (Velban®) were the first plant- derived anticancer agents to be approved by the FDA, in 1963 and 1965, respectively
(FDA, 1963; FDA, 1965; Guérritte and Fahy, 2005). Currently, vincristine sulfate is in 3 clinical use to treat acute lymphoblastic leukemia and certain lymphomas in combination chemotherapy (Chabner et al., 2006). Vinblastine sulfate is used for the treatment of
Hodgkin’s lymphoma and for bladder cancer and some breast cancer types (Cragg and
Newman, 2005; Chabner et al., 2006). The structurally related vinca alkaloids, vindesine
(3) and vinorelbine (4), are semisynthetic derivatives of vinblastine. Vindesine is being used in the treatment of Hodgkin’s and non-Hodgkin’s lymphomas in France and the
U.K., but has not been approved in the U.S. for the treatment of these conditions (Dancey and Steward, 1995). Vinorelbine tartrate, however, has been approved for the treatment of non-small cell lung carcinomas (NSCLC) in the United States (Brogden and Nevidjon,
1995).
2. Epipodophyllotoxin derivatives
Etoposide (5) and teniposide (6) are semi-synthetic derivatives of podophyllotoxin (7), which was isolated initially from Podophyllum peltatum L.
(Berberidaceae) (American mandrake). The first isolation report of podophyllotoxin appeared in 1880, but the structure was not elucidated until 1951 by Hartwell and
Schrecker (Hartwell and Schrecker, 1951). Etoposide and teniposide interfere with topoisomerase-mediated re-annealing of single- and double- strand DNA breaks in the S and G2 phases of the cell cycle (van Maansen et al., 1988). This results in accumulation of DNA damage and potent induction of caspase-dependent apoptosis. Due to the low water solubility of etoposide, large fluid volumes are needed to achieve a therapeutically effective dose, which can cause heart failure, hypersensitivity reactions and hyptotension
(Hande, 1998). The pro-drug, etoposide phosphate, has overcome these solubility issues, 4 and is converted in the body to the active drug etoposide by alkaline phosphatases, and does not show toxicities beyond those of etoposide (Hande, 1998). Etoposide phosphate is used clinically for the treatment of lung and testicular cancers as well as for leukemias and lymphomas (Stadtmauer et al., 1989; Carney et al., 1990; Loehrer, 1991; Keane and
Carney, 1993). Teniposide has clinical use as a front-line treatment for childhood acute lymphoblastic leukemia (ALL), as well as refractory adult ALL and acute monoblastic leukemia (Sonneveld, 1992).
3. Taxanes
The taxanes are an important class of cancer chemotherapeutic agents. Paclitaxel
(Taxol®) (8) was isolated from the bark of the Pacific Yew tree, Taxus brevifolia Nutt.
(Taxaceae) and structurally characterized by Wall and Wani in 1971 (Wani et al., 1971).
Initial bioactivity-guided fractionation of extracts from T. brevifolia was assayed using in vitro 9KB and P388 leukemia cells, with 8 shown to be active for mice bearing L1210 leukemic cells (Wall and Wani, 1996). Later studies on the mechanism of action of 8 revealed that unlike the vinca alkaloids, which inhibit the polymerization of tubulin to form microtubules, paclitaxel stabilizes microtubules and inhibits depolymerization back to tubulin (Schiff et al., 1979; Horwitz, 1992). Its novel chemical structure combined with a unique mechanism of action propelled paclitaxel through clinical development, and ultimately led to FDA approval for the treatment of head, neck and ovarian cancer
(Slichenmyer and von Hoff, 1991; Cragg and Newman, 2005). The success as a lead compound of the unmodified natural product, paclitaxel, is apparent considering there were nearly 25 additional taxanes in preclinical development as potential anticancer 5
agents a few years ago (Cragg and Newman, 2005). Two semi-synthetic taxane
derivatives, docetaxel (Taxotere®) (9) and cabazitaxel (Jevtana®) (10), have also gained
market approval. Docetaxel was FDA-approved in 2006 for the treatment of patients with
advanced squamous cell carcinoma of the head and neck (FDA, 2006). Cabazitaxel was
approved by the FDA in 2010 for the treatment of patients with metastatic hormone-
refractory prostate cancer (FDA, 2010). In particular, cabazitaxel is gaining use for
patients having a variety of docetaxel-refractory tumors. A phase III trial in 775 men with
metastatic castration-resistant prostate cancer comparing prednisone plus 12 mg/m2 mitoxanthrone i.v. over 15-30 min or prednisone plus 25 mg/m2 cabazitaxel i.v. over 1 h
every 3 weeks was conducted. The study found that median progression-free survival was
2-8 months in the cabazitaxel group and 1-4 months in the mitoxantrone group, thus
carbazitaxel improved overall survival (de Bono et al. 2010, Pal et al. 2010).
4. Camptothecins
The isolation and structure elucidation of the antitumor lead compound
camptothecin (11) from Camptotheca acuminata Decne. (Cornaceae) was first reported
in 1966 by Wall and Wani (Wall et al., 1966). The intial antitumor activity can be
attributed to early testing of extracts from C. acuminata cytotoxicity towards 9KB cells,
and a L1210 mouse leukemia life prolongation in vivo assay, which revealed marked
activity with doses as low as 0.5 mg/kg (Wall and Wani, 1996). The low water solubility
of the parent compound camptothecin was the impetus for administration of its sodium
salt, which showed efficacy in a number of clinical trials. In a phase I clinical trial
involving 18 patients with gastrointestinal tumors, it was found that upon administration 6 of camptothecin, partial responses were observed in five patients (Gottlieb and Luce,
1972). In a Chinese clinical trial involving 1000 patients, the sodium salt of campothecin showed efficacy against gastric and intestinal cancers, as well as head and neck tumors, and bladder carcinoma (Xu et al., 1980). Despite showing promise in clinical trials of campothecin sodium salt between the 1970s and 1980s, the compound was dropped in the the U.S. due to bladder toxicity (Cragg and Newman, 2005). The semisynthetic analogues, irinotecan (Camptosar®) (12) and topotecan (Hycamtin®) (13), however, show less toxicity and were introduced successfully into therapy as clinical agents. Irinotecan is used in combination with fluorouracil and leucovorin as a therapy for patients with metastatic colorectal cancer (Saltz et al., 2000). Topotecan has clinical use in the treatment of small cell lung cancer (SCLC) (Brahmer and Ettinger, 1998). In addition,
FDA approval was gained for the use of topotecan in patients with stage IVB recurrent or persistent carcinoma of the cervix that is not amenable to curative treatment with surgery and/or radiation therapy (Brave et al., 2006). It has been estimated that from 2006-2008, nearly $4 billion was spent on cancer chemothearapy using irinotecan and topotecan
(Cragg et al., 2009). Moreover, the promise for the clinical introduction of other compounds based on the camptothecin skeleton is clear, with over 20 such derivatives being in clinical trials recently (Cragg et al., 2009).
5. Omecetaxine mepesuccinate (homoharringtonine)
Omacetaxine mepesuccinate, (Synribo®) (14) was approved by the U.S. FDA in
2012 for the treatment of chronic myelogenous leukemia (FDA, 2012). This compound, known earlier as homoharringtonine was isolated over 40 years ago from Cephalotaxus 7 harringtonia Knight ex J. Forbes K. Koch (Cephalotaxaceae) by Powell and Smith
(Powell et al., 1972), and is an ester of the parent compound, cephalotaxine, first reported by Paudler and associates in the early 1960s (Paudler et al., 1963). Homoharringtonine functions as a protein synthesis inhibitor, blocking peptide bond formation and aminoacyl tRNA binding, thus inhibiting translation at the elongation phase (Tujebajeva et al.,
1989). In an in vitro assay, homoharringtonine alone or in combination with IFN- and cytosine arabinoside (Ara-C), induced apoptosis in human normal and chronic myelogenous leukemia (CML) hematopoietic progenitor cells at concentrations as low as
100 ng/ml (Visani et al., 1997). Another study investigating the effects on clonal proliferation and differentiation found that homoharringtonine inhibited colony formation of myeloid and lymphoid cell lines and fresh leukemic cells (Zhou et al., 1990). In a clinical trial with this compound involving patients with late-stage chronic myelogenous leukemia (CML), 72% of the 58 evaluable patients exhibited a hematological response
(O’Brien et al., 1995). In a separate trial, the same 58 patients treated with homoharringtonine were compared to a group of patients having only had IFN- alone.
The results showed that in 92% of patients, a complete hematological response was observed, which was much improved from patients receiving only IFN- (O’Brien et al.,
1995).
6. Trastuzumab emtansine
The parent compound of the cytotoxic maytansinoid class is maytansine (15), an ansa macrocycle isolated in 1972 by Kupchan and colleagues from the Ethiopian shrub
Maytenus ovatus Wall. ex Wight & Arn. (Celastraceae) (Kupchan et al. 1972). 8
Maytansinoids are antimitotic agents that bind tubulin, resulting in inhibition of microtubule assembly (Remillard et al., 1975; Wolpert-Defilippes et al. 1975). In vivo studies using the Lewis lung carcinoma B-16 melanocarcinoma murine solid tumor model, and against P-388 lymphocytic leukemia, showed activity over a 50-100 fold range at the µg/kg level. Conjugation of a number of maytansinoids with monoclonal antibodies has shown the promise of increased compound targeting to cancerous cells, and lower side effects (Ducry and Stump, 2010). The maytansinoid conjugate, trastuzumab emtansine (T-MD1, Kadcyla®) (16), comprised of the antibody trastuzumab, the maytansinoid DM1 (mertansine), and a linker, was approved by the U.S. FDA in
February 2013 for the treatment of HER2-positive metastatic breast cancer (Verma et al.
2012; FDA, 2013).
7. Ingenol mebutate
Plants in the genus Euphorbia (Euphorbaceae) have been used in traditional medicine to treat malignancies and warts (Hartwell, 1971). Species in this genus are well- known to contain skin-irritant compounds, particularly tigliane, ingenane and daphnane esters (Kinghorn, 1979). Bioassay-guided fractionation of the stem wood of Euphorbia quinquecostata using a phorbol dibutyrate receptor-binding assay yielded compounds with activities in the low micromolar range (Mbwambo et al., 1996). Compounds from E. pubescens were investigated for multidrug resistance (MDR) reversing activity on mouse lymphoma cells. It was found that a jatrophane-type diterpene polyester, pubescene D, exhibited strong activity comparable to the positive control verapamil (Valente et al.,
2004). Ingenol mebutate (ingenol 3-angelate) (17), a constituent of the plant, Euphorbia 9 peplus L., was approved by the U.S. FDA in 2012 for the treatment of actinic keratosis, a precursor condition to sun-related squamous cell carcinoma (Lebwohl et al., 2012). The treatment of actinic keratosis and the surrounding skin can ameliorate clinical symptoms.
Topical gel formulations consisting of 0.015% and 0.05% ingenol mebutate for the face, scalp, and extremeties are in clinical use (Lebwohl et al., 2012). Ingenol 3-angelate was first isolated from Euphorbia peplus and characterized by Hohmann et al. (Hohmann et al., 2000).
Vinblastine (1) Vincristine (2) Vindesine (3)
Vinorelbine (4) Etoposide (5) Teniposide (6)
Podophyllotoxin (7) Etoposide phosphate (8) Figure 1.1. Structures of plant-derived anticancer drugs currently on the market and selected related compounds (continued on page 11).
10
Figure 1.1 (continued). Structures of plant-derived anticancer drugs currently on the market and selected related compounds.
Paclitaxel (9) Docetaxel (10) Cabazitaxel (11)
Camptothecin (12) Irinotecan (13)
Topotecan (14) Omeacetaxine mepesuccinate (15)
Trastuzumab emtansine (16) Ingenol mebutate (17)
11
Code Trivial name Source Clinical Use Reference 1 vinblastine Catharanthus roseus Hodgkin’s Guérritte, 2005; lymphoma; non- Chabner, 2006 small cell lung carcinoma (NSCLC); breast cancer; bladder cancer 2 vincristine Catharanthus roseus Lymphoblastic Guérritte, 2005; leukemia; Chabner, 2006 lymphoma 3 vindesine semi-synthetic non-Hodgkin’s Dancey and lymphoma, Steward, 1995 Hodgkin’s lymphoma 4 vinorelbine semi-synthetic non-small cell Brogden and lung carcinoma Nevidjon, 1995; (NSCLC) 5 etoposide semi-synthetic Lung, testicular Hande, 1998 cancer, leukemia, lymphoma 6 teniposide semi-synthetic Acute Hande, 1998 lymphoblastic leukemia (ALL) in children, refractory ALL, acute monoblastic leukemia 7 etoposide semi-synthetic prodrug lung and testicular Stadtmauer et al., phosphate cancer, leukemias 1989; Carney et and lymphomas al., 1990; Loehrer, 1991 9 paclitaxel Taxus brevifolia; semi- Head, neck, and McGuire et al., synthesis; plant tissue ovarian cancer 1996; culture Slichenmyer and von Hoff, 1991 10 docetaxel semi-synthesis; Advanced FDA, 2006 synthesis squamous cell carcinoma of the head and neck
Table 1.1. Plant-derived anticancer compounds currently on the market (continued on page 13).
12
Table 1.1 (continued). Plant-derived anti-cancer compounds currently on the market. Code Trivial name Source Clinical Use Reference 11 cabazitaxel semi-synthesis; Metastatic de Bono et al., synthesis hormone- 2010, Pal et al., refractory 2010 prostate cancer 13 irinotecan semi-synthesis Metastatic Saltz et al., 2000 colorectal cancer 14 topotecan semi-synthesis Small cell lung Brahmer and cancer, stage Ettinger, 1998; IVB recurrent or persistent carcinoma of the cervix 15 omacetaxine Cephalotaxus Chronic FDA, 2012 mepesuccinate harringtonia myelogenous leukemia (CML) 16 trastuzumab Antibody-drug HER2-positive Verma et al., 2012 emtansine conjugate; metastatic Maytenus ovatus breast cancer
B. Nicotinic Acetylcholine Receptor (nAChR) Agents from Natural Sources
Natural products have been an important source of drugs to treat disorders of the central nervous system (Clement et al., 2004). Nicotinic acetylcholine receptors
(nAChRs) are linked to several physiological functions and disease states, so the development of drugs that have such activity is of particular interest. Physiological functions attributed to nAChRs affect the memory, learning, attention, pain perception, and body temperature regulation (Cooper et al., 1991; Lukas et al., 1999; Lloyd and
Williams, 2000; Jensen et al., 2005). Influence on the release of acetylcholine (ACh) implicates nAChRs in the modulation of neurotransmitter levels, particularly of dopamine, norepinephrine, serotonin, glutamate and -aminobutyric acid (Jensen et al.,
13
2005). In addition to a role in cholinergic signaling, nAChRs influence many other neurotransmitter systems of the CNS and hence are implicated in a wide variety of disease states such as nicotine addiction, epilepsy, Parkinson’s disease, Alzheimer’s disease, Tourette’s syndrome, schizophrenia, anxiety, and depression (Lukas et al., 1999;
Lloyd and Williams, 2000; Jensen et al., 2005; Dani and Bertrand, 2007; González-
Cestari et al., 2009).
Structurally, nAChRs are pentameric ligand-gated ion channels expressed in the plasma membrane of all mammalian cells (Cooper et al., 1991). They allow the influx of cations such as Na+, K+, and Ca2+ (Jensen et al., 2005). There are two varieties of nAChRs. The first of these is the muscle-type located postsynaptically at neuromuscular junctions that contribute to skeletal muscle movement. The second form is the neuronal type, located both presynaptically and postsynaptically in autonomic ganglia and in cholinergic neurons (Jensen et al., 2005). There are 17 nAChR subunits organized by anatomical distribution, with muscle-located subunits named 1, 1, , , , and neuronal subunits named 2- 10 and 2- 4 (Jensen et al., 2005). The native agonist acetylcholine binds at the interface between the and subunits (Dani and Bertrand, 2007). The nomenclature for nAChRs reflects the large amount of possible subunit combinations, with nAChRs composed of identical 7, 8, or 9 (homomeric), or heteromeric combinations such as 4 2 (Lukas et al., 1999). It is the large variety of possible subunit combinations that gives rise to a variety of physiological roles. Each subunit of a nAChR has four transmembrane spanning regions with extracellularly located N and C termini
(Cooper et al., 1991; Lukas et al., 1999; Jensen et al., 2005) (Figure 1.2). Figure 1.2 is
14 based on the acetylcholine-binding protein (AChBP) found in the water snail, Lymnaea stagnalis. The AChBP has been a popular model system for nAChRs because of homologous structural features shared with nAChRs such as the cysteine-loop of the N- terminal domain (NTD), the formation of pentameric complexes, and an affinity to a wide range of cholinergic ligands (Jensen et al., 2005).
L
N helix
L3 L N-Terminal L6 domain L (NTD) -helices
Cys L2 L9 Hallmark "Cys loop" between 6 and 7 Cys shown in purple. nAChRs L7 C are sometimes called "cys L5 loop"receptors due to this structural feature (Celie et Extracellular al.,2005).
Ion-channel domain (ICD) M4 M3 M2 M1
Intracellular
Figure 1.2. Topology of the nAChR subunits. The two regions involved in the formation of the N-terminal domain (NTD) and the ion-channel domain (ICD) of the pentameric nAChR are shown. The 10 -strands and the loops between in the N-terminal domain are numbered according to the AChBP structure. The disulfide bond between two cysteines creates the hallmark “Cys-loop” between 6 and 7.
15
Conserved amino acids in the ion channel of nAChRs are important for ion
permeability. Calcium permeability of the 7 nAChR, for example, is dependent on the
arrangement of amino acid residues in the pore (Dani and Bertrand, 2007). Membranes
from Torpedo marmorata were examined using electron microscopy, and it was shown
that the ion channel of the Torpedo nAChR is 40 Å long with a diameter of 80 Å (Unwin,
1995; Miyazawa et al., 2003) (Figure 1.3). In the resting state, aliphatic hydrophobic
amino acids (Val, Leu, Ile) are situated at the portion of the ion channel contained within
the plasma membrane, in the so-called “resting gate” state of the receptor (Unwin, 1995;
Miyazawa et al., 2003). The ion channel width at the “resting gate” is only 6 Å wide,
making the permeation of hydrated ions impossible (Jensen et al., 2005). In addition,
negatively charged residues appear at the intracellular and extracellular ends of the ion
pore (Miyazawa et al., 2003). The reliance of ion conductance on the sequence of amino
acids in the nAChR pore was shown in a study that mutated a glutamate residue at the
inner mouth of the pore to a neutral alanine, resulting in suppressed calcium permeability
(Bertrand et al., 1993; Jensen et al., 2005) (Figure 1.3). The nature of the charged ion
passing through the pore can also be changed selectively, if the amino acid sequence of
the ion pore changes. A mutation of Glu 241 to Ala completely abolishes Ca2+ permeability (Bertrand et al., 1993; Jensen et al., 2005).
16
80 Å
6 Å 40 Å
Figure 1.3. (Top) nAChR residues lining the ion pore in Torpedo species, which are conserved in most neuronal nAChR subunits. The localization of the negatively charged residues at the mouth of the ion channel, the ion charge selectivity filter, and the resting gates are shown. (Bottom) Sequence alignment of the 1, 4, 7 subunits in Torpedo nAChR, with charged residues and resting gates in the receptor boxed (adapted from Miyazawa et al., 2003; Jensen et al., 2005).
17
In the resting state, the plasma membrane carries a positive charge on the outside,
negative on the inside. When the agonist nicotine binds, conformational changes in the
receptor permit Ca2+ to flow into the cell, depolarizing the membrane (Schuller, 2009).
The change in charge causes the gates of separate voltage-gated Ca2+ channels to open,
leading to more Ca2+ influx. This sequence of events in nAChR activation has been
examined using dose-responses of specific subtypes to endogenous ACh and nicotine. In
one study employing patch-clamping in K-177 cells expressing h 4 2 and 7 receptors,
long-term exposure to nicotine was investigated (Buisson and Bertrand, 2001). It was
shown that 7 nAChRs have a low affinity for ACh activation, with an ED50 of 200 µM,
but h 4 2 nAChRs have much higher affinity for ACh activation, with typical ED50 values as low as 1.6 µM (Buisson and Bertrand, 2001). Overnight exposure of the K-177 cells to 100 nM-10 µM nicotine revealed that patch-clamp time-response curves were slower, with a decline in the current carried by the nAChR (Buisson and Bertrand, 2001).
The authors suggested, as a result of these experiments, that nicotine agonists cause receptor desensitization, characterized by a decline in the nAChR current, ultimately leading to closure of the receptor.
1. Drug discovery approches related to nAChR action
A high degree of sequence homology among nAChR and subunits in the ligand binding domain for nAChRs makes development of drugs that specifically target receptor subtypes difficult (Jensen et al., 2005; González-Cestari et al., 2009). In particular, the amino acid sequences encoding the proteins that form orthosteric sites, such as the ACh binding site, have a high degree of sequence identity (Henderson et al., 18
2010). To develop drugs with nAChR subtype selectivity, an alternative approach is to target allosteric sites, which are defined as compound binding sites distinct from the orthosteric site that modulate receptor function (González-Cestari et al., 2009; Henderson et al., 2010).
2. Natural sources of nAChR agonists and antagonists
Some natural products with activity at nAChRs serve as pharmacological probes.
Owing to the structural complexity of isolated natural products, historically they have comprised nAChR ligands with activity targeted specifically at nAChRs, rarely contributing cross-activity affecting other receptors (e.g., 5-HT serotonin, NMDA)
(Jensen et al., 2005). Due to the several possible subunit combinations in neuronal nAChRs, subtype selective natural products are less common. The prototype ligand for nAChRs is (-)-nicotine (18), with the S enantiomer having the most potent activity at nAChRs (Bunnelle et al., 2004).
2.1 (-)-Cytisine
The quinolizidine alkaloid, (-)-cytisine (19), exhibits selective agonist activity for neuronal nAChRs over muscle-type nAChRs, which is not seen for either nicotine or epibatidine (Romanelli and Gualtieri, 2003; Bunnelle et al., 2004; Jensen et al., 2005). Its occurrence in plants has been thoroughly investigated for over 100 years, having been reported in several legume genera including Anagyris, Baptisia, Euchresta, Genista,
Sophora, Thermopsis and Ulex (Henry, 1949a). Cytisine was isolated by Partheil in 1894, with Ing and Cahn also contributing to the structural proof via synthetic degradation
19
studies (Partheil, 1984; Ing and Cahn, 1931). The toxicity of (-)-cytisine has been
established for many decades, with reports in the early 1900’s describing symptoms
including nausea, convulsions, and death by failure of respiration (Henry, 1949a).
Compared to nicotine, cytisine is a partial agonist at nAChRs with subnanomolar affinity
for the 4 2 subtype, and is a more potent ganglionic stimulant (Bunnelle et al., 2004).
The total synthesis of the compound has been achieved, with the goal of furthering the
therapeutic potential in the treatment of nicotine addiction (O’Neill et al., 2000).
2.2 Lobeline
The genus Lobelia was first shown to contain alkaloids in the late 19th century
(Henry, 1949b). The piperidine alkaloid lobeline (20) was isolated initially as
“lobelidine” in 1921 by Bastick and later shown by Wieland, Koschara and Dane to be dl-lobeline (Wieland et al., 1929). Lobeline is a component of Lobelia inflata L.
(Campanulaceae) (Indian tobacco) (Bunnelle et al., 2004), and has similar pharmacological effects to nicotine such as brachycardia and hypotension. However, the effects are not blocked by mecamylamine, suggesting an alternative mechanism of action
(Brioni et al., 1993). The compound has high affinity at ganglionic nAChR subtypes with a Ki of 4 nM (Badio et al., 1995). Lobeline may effect inhibition of dopamine uptake and
stimulate dopamine release from synaptic vesicles (Dwoskin and Crooks, 2002).
Applications to smoking cessation and treatment of CNS disorders are current areas of
investigation of which lobeline is representative (Crooks and Dwoskin, 1998; Eswara et
al., 1998).
20
2.3 Galanthamine and Physostigmine
Other allosteric modulators include galanthamine (21) and physostigmine (22).
Both bind allosterically in the N-terminal domain (NTD) of the -subunit of the nAChR, increasing the receptor affinity for an orthosteric agonist (Pereira et al., 1993;
Schrattenholz et al., 1996; Pereira et al., 2002; Dajas-Bailador et al., 2003; Jensen et al.,
2005). Galanthamine was isolated by Mashkovsky and Kruglikova in 1951 from
Galanthus woronowii Losinsk. (Amaryllidaceae) (Mashkovsky and Kruglikova-Lvova,
1951). Galanthamine has been shown to potentiate the effects of nAChR agonists on norepinephrine, dopamine and GABA release in the brain (Pereira et al., 2002, Dajas-
Bailador et al., 2003; Jensen et al., 2005).
Physostigmine was isolated about 150 years ago from the seeds of Physostigma venenosum Balf. (Leguminosae), in an amphorous condition (Jobst and Hesse, 1864).
The first crystalline form was isolated by Vee and Leven and was called eserine (Vee and
Leven, 1865). The structure was later corrected by Julian and Pikl through chemical synthesis (Julian and Pikl, 1935). Physostigmine functions as an acetylcholinesterase drug and is used clinically as a treatment for curare poisoning and glaucoma (Triggle et al., 1998). Physostigmine has been shown to densitize, block or act as an agonist at nAChRs (Katz and Miledi, 1977; Sherby et al., 1984; Pereira et al., 1993).
2.4 D-Tubocurarine
The tetraisoquinoline alkaloid D-tubocurarine (23) was isolated from the South
American plant, Chondrodendron tomentosum Ruiz & Pav. (Menispermaceae), by King in 1935, who was the first to obtain crystals (King, 1935). The compound was also 21
isolated and described by Dutcher who corroborated the structure (Dutcher, 1946). D-
Tubocurarine is a less discriminative nAChR receptor antagonist showing competitive antagonistic activity that inhibits depolarization in postsynaptic nerve terminals (Jensen et al., 2005). A classic study analyzing the potency of D-tubocurarine with other antagonists
in the soleus muscle in cats under anesthesia found that D-tubocurarine was five times
more potent at blocking contractions of the soleus muscle than pancuronium (Bowman
and Webb, 1972).
2.5 Epibatidine
Epibatidine (24) is a potent non-opioid, nonselective nAChR agonist, isolated
from the poison dart frog Epipedobates anthonyi Noble (formerly Epipedobates tricolor
Boulenger (Dendrobatidae), which was first reported in 1992 by Daly and associates
(Spande et al., 1992; Badio and Daly, 1994). In the initial 1992 isolation, 750 frogs were
required to produce an alkaloidal extract of 60 mg, to facilitate isolation work that
ultimately yielded epibatidine in trace amounts (0.75 mg; about 1 µg per frog) (Spande et
al., 1992). The bioactivity-guided fractionation was monitored by Straub-tail equivalents
in mice, with an erect tail signifying the administration of an nAChR agonist (Spande et
al., 1992). A comparison of the activity of epibatidine to morphine showed epibatidine to
be 200 times more potent than morphine at producing analgesia in the mice hot plate test
(e.g., 1 mg/kg morphine; 0.005 mg/kg epibatidine). In the Straub-tail reaction test, the tail
arch elicited by morphine was blocked by administration of 5 mg/kg naloxone 20 min
prior to the administration of 20 mg/kg morphine. The Straub tail test showed that only
20 µg/kg epibatidine caused the same tail reaction as morphine (10 mg/kg), and was only 22 slightly reduced by naloxone, with the tail-arch persisting for an additional 1-2 h (Spande et al., 1992). The natural (+)-epibatidine gave an ED50 value of 1.5 µg/kg (i.p.) in mice, and showed little or no activity at opioid, muscarinic, adrenergic, dopamine, serotonin, and -aminobutyric acid receptors (Spande et al., 1992). Synthetic analogues of epibatidine have been produced to combat the lack of nAChR subtype selectivity and poor therapeutic index of the naturally occurring alkaloid (Badio et al., 1995; Bunnelle et al., 2004).
2.6 Anabaseine
Anabaseine (25) has been shown to be present in the marine worm Paranemetres peregrine (Emplectonematidae) and in Aphaenogaster (Formicidae) ant species (Kem,
1971; Wheeler et al., 1981; Asakawa et al., 2013). A structurally similar compound, anabasine, was isolated much earlier from the plant Anabasis aphylla L.
(Chenopodiaceae), in 1929 (Orekhov, 1929). In hoplonemertine carnivorous worms, anabaseine is released from the proboscis as it feeds on other annelids, paralyzing the prey (Kem, 1971). Pharmacologically, anabaseine is a nonselective, weak partial nAChR agonist at the 4 2 subtype, but has shown higher efficacy at the 7 nAChR receptor subtypes (Kem, 1971; De Fiebre et al., 1995; Jensen et al., 2005). Owing to its 7- selective potency, many synthetic analogues have been produced, based on anabaseine, with the aim of affording novel selective agonists (Bunnelle et al., 2004). The affinities
(Ki values) for anabaseine, anabasine and nicotine at rat brain 7 receptors were shown to be 0.058 ± 0.007 µM, 0.058 ± 0.028 µM, and 0.40 ± 0.035 µM, respectively, when expressed in terms of their active monocation concentrations (Kem et al., 1997). 23
2.7 Ivermectin
Ivermectin (26), a semi-synthetic derivative of avermectin isolated from the soil bacterium Streptomyces aermitilis (Streptomycetaceae), is an anthelmintic (Burg et al.,
1979). It was determined that preapplication of micromolar concentrations of ivermectin increased the current evoked by subsequent application of ACh in neuronal chick or human 7 nAChRs reconstituted in Xenopus laevis oocytes and K-28 cells, increasing the affinity for ACh by 20-fold (Krause et al., 1998). It was concluded that ivermectin acts on the 7 nAChR as a positive allosteric modulator (Krause et al., 1998).
2.8 -Bungarotoxin and -Conotoxins
Other than small-molecule nAChR agonists and antagonists, peptide compounds with such activity have also been isolated and studied, particularly -bungarotoxin from the Taiwan banded krait Bungarus multicinctus (Elapidae) and -conotoxin from the
Conus genus (Conidae) of marine snails. -Bungarotoxin is part of large family of toxins, differing in peptide sequence, with -bungarotoxin being highly studied due to its competitive antagonism at 7 nAChRs (Moise et al., 2002; Jensen et al., 2005). At low doses, -bungarotoxin was found to selectively block the 3 4 receptor expressed in
Xenopus oocytes (Colquhoun and Patrick, 1997).
The -conotoxins are venoms used by predatory cone snails and are just a subsection of the nearly 50,000 toxins produced by these organisms (Olivera et al.,
1990). -Conotoxins, as opposed to A, and K structural varieties, are the only type known to act on neuronal nAChRs (Jensen et al., 2005). They possess four cysteine residues, having disulfide bonds between both the first and third residues and the second 24 and fourth cysteine residues. Unlike -bungarotoxin, -conotoxins do not antagonize muscle-type nAChRs (Jensen et al., 2005). Depending on the sequence of amino acids in the conotoxin, differences in the selectivity profile and IC50 values are observed (Nicke et al., 2003; Loughnan et al., 2004).
Ziconotide (27), a drug from the peptide class of nAChR active natural products is indicated for the treatment of chronic pain, and was approved by the U.S. FDA in 2004
(Celie et al., 2005). Ziconotide, produced by chemical synthesis, is a competitive nAChR inhibitor consisting of two helical amino acid sequence folds, braced by two conserved disulfide bonds forming a two-loop framework (Celie et al., 2005).
(-)-cytisine (19) lobeline (20) (-)-nicotine (18)
galanthamine (21) physostigmine (22)
Figure 1.4. Structures of selected compounds with activity at nicotinic acetylcholine receptors (nAChRs) (continued on page 26).
25
Figure 1.4 (continued). Structures of selected compounds with activity at nicotinic acetylcholine receptors (nAChRs).
epibatidine (81) anabaseine (25) D-tubocurarine (23)
ivermectin (26)
Ziconotide (27)
26
Compound Source Pharmacological Effect References (-)-nicotine (18) Nicotiana tabacum Prototype nAChR agonist, mood-alterant, Romanelli and Gualtieri, 2003; stimulant, relaxant Jenson et al., 2005 (-)-cytisine (19) Anagyris, Baptisia, partial agonist at nAChRs with Papke and Heinemann, 1994; Euchresta, Genista, Sophora, subnanomolar affinity for the 4 2 subtype; O’Neill et al., 2000; Bunnelle et Thermopsis,Ulex spp. potent ganglionic stimulant al., 2004 lobeline (20) Lobelia inflata inhibition of dompamine uptake and Crooks and Dwoskin, 1998; stimulate dopamine release from synaptic Eswara et al., 1998; Dwoskin and vesicles Crooks, 2002 galanthamine (21) Galanthus woronowii potentiates the effects of nAChR agonists Pereira et al., 2002; Dajas- on norepinephrine, dopamine and GABA Bailador et al., 2003; Jensen et release in the brain al., 2005 physostigmine (22) Physostigma venenosum acetylcholinesterase drug used as a Katz and Miledi, 1977; Sherby et treatment for curare poisoning and al., 1984; Pereira et al., 1993; glaucoma; densitizes, blocks or acts as an Triggle et al., 1998
27 agonist at nAChRs D-tubocurarine (23) Chondrodendron nAChR receptor antagonist; competitive Bowman et al., 1972; Jensen et tomentosum antagonistic activity; inhibits depolarization al., 2005 in postsynaptic nerve terminals epibatidine (24) Epipedobates anthonyi potent non-opioid, nonselective nAChR Badio and Daly, 1994; Spande et agonist al., 1992 anabaseine (25) Paranemetres peregrina; nonselective, partial nAChR agonist at Kem et al., 1997; De Fiebre et al., Aphaenogaster ant species 4 2 subtypes; higher efficacy at 7 1995; Jensen et al., 2005 nAChR receptor subtypes ivermectin (26) Streptomyces aermitilis 7 nAChR positive allosteric modulator Krause et al., 1998 ziconotide (27) Synthetic version of natural competitive nAChR inhibitor; chronic pain Celie et al., 2005 product relief
Table 1.2 Natural products with activity at nicotinic acetylcholine receptors (nAChRs). 27
Chapter 2: Taxonomy and Phytochemistry of Microcos paniculata
A. Background on Microcos
1. The family Malvaceae
The family Malvaceae, or by its common name, the Mallow family, is distributed mostly in tropical climates (e.g., Africa, Latin America, South Asia) and some temperate climatic zones (Stephens, 2012). The family contains approximately 204 genera and 2330 species, with the genus Hibiscus (300 spp.) being the largest (Judd, 2002). Several species in this family have economic importance, such as the food plants Theobroma cacao (chocolate), Cola acuminata (cola seeds), and Hibiscus esculentus (okra), as well as Ochroma pyramidala (balsa wood, a timber product), Gossypium species (cotton, fabrics), and Althaea species (hollyhock, ornamentals) (Judd, 2002). A botanical description of Malvaceae is provided as follows (Ahles, 1968; Judd, 2002; Stephens,
2012):
Habit and duration. Trees, shrubs, lianas, or herbs. Annual or perennial with cosmopolitan distribution. Leaf anatomy. Leaves alternate, simple, unlobed or dissected, usually palmately; stipules present, often caducous. Stellate pubescence is common throughout the family. Flower anatomy. Flowers axillary, racemose or paniculate, each flower often pedunculate and pedicellate; involucre present or absent. Flowers
28
bisexual or unisexual, usually radial, often associated with conspicuous bracts that form an epicalyx. Sepals 5, united at base or more than 1/2 the length; petals 5, united at base, rounded or emarginate at apex, often very showy; stamens usually numerous, united to form a long tube (staminal column) surrounding the ovary and styles, filaments separate near the apex, each with a single 1-locular anther. Pistil 1, ovary superior; stigmas capitate or filamentous, as many as the carpels or 2x as many. Fruit anatomy. Capsule (often forming a ring of carpels, usually separating from one another and from the axis), rarely a berry or samara. Nectaries composed of densely packed, multicelluar, glandular hairs on sepals and sometimes on petals or androgynophore. Fruit usually a loculicidal capsule, schizocarp, nut, indehiscent pod, aggregate of follicles, drupe, or berry; seeds sometimes with hairs or arillate, occasionally winged; embryo straight to curved; endosperm present. Vasculature. Cork cambium outer cortical, pits not vestured; tile cells common; sieve tubes with non-dispersive protein bodies.
2. Nomenclature and classification for the genus Microcos and the closely related genus Grewia
The genus Microcos is closely identified with the genus Grewia, which has resulted in changes in nomenclature. In 1767, Linnaeus combined the two genera, accepting Grewia L. as the correct name for the combined taxon; relegating Microcos L. to become a subgenus of Grewia L. However, since the name Microcos paniculata L. had been previously published by Linnaeus in 1753, Grewia paniculata L. was deemed a superfluous name (Panigrahi, 1985). Linnaean names later discovered to be superfluous by modern botanists are matters of nomenclature, which has strict rules about the name that must apply in a given situation. However, such nomenclature does not dictate classification and each botanist is free to choose the classification to follow (Judd, 2002).
The modern Angiosperm Phylogeny Group III (APG III) system organizes plants based
29 on similarities in DNA sequences with phylogenetic trees, correcting ambiguities in earlier Linnean groupings (Judd, 2002). When applied to a field specimen chosen to be classified within Microcos, nomenclature dictates that the specimen be called Microcos paniculata L. If considered part of Grewia, the field specimen should be called Grewia nervosa (Lour.) Pangr. (Panigrahi, 1985). Morphological characteristics of the collected field specimens were compared with identified specimens of Grewia and Microcos and named Microcos paniculata L., utilizing Flora of China as the floristic authority (Ya et al., 2007).
3. Genus Microcos and Microcos paniculata L.
The genus Microcos contains about 60 species distributed primarily in Africa and
Asia (Ya et al., 2007). Ethnomedical uses of the genus, particularly Microcos paniculata
L., include utilization as herbal teas for the treatment of colds, enteritis, and skin rashes in southern mainland China (Ya et al., 2007; Feng et al., 2008a). In the discussion of the nomenclature and classification for Microcos and Grewia in section A.2 above, it was stated that Linnaeus classified Microcos as a subgenus of Grewia. Indeed, close morphological characteristics unify the two genera, such as a calyx with free sepals, drupe-type fruits that are often 2- or 4-lobed, and a short androgynophore (Ya et al.,
2007). However, close inspection of the two genera has shown that the inflorescences in
Grewia are cymose, axillary, with a swollen, lobed stigma (Ya et al., 2007). In contrast, in the genus Microcos, the inflorescence is paniculate and terminal, with a subulate, unlobed stigma (Ya et al., 2007). The genus Microcos has been described as follows (Ya et al., 2007): 30
Habit and duration. Shrubs or small trees. Primarily in Africa, Asia; three species in China. Leaf anatomy. Leaves alternate, shortly petiolate; leaf blade ovate, oblong, or lanceolate, leathery, basal veins 3, margin entire or lobed in distal 1/2. Inflorescence. Terminal or axillary, cymose paniculate. Flower anatomy. Bisexual. Sepals 5, free. Petals 5 or rarely absent, glandular at base adaxially. Stamens many, free, borne distally on androgynophore. Ovary superior, usually 3-loculed; ovules 4-7 per locule; style simple; stigma subulate, usually not lobed. Fruit anatomy. A drupe, globose, obovoid, or pyriform, not furrowed, without drupelets.
In turn, Microcos paniculata L. has been described as follows (Ya et al., 2007)
Habit and duration. Shrubs or small trees 3-12 m tall. Bark rough; branchlets hairy. Leaf anatomy. Stipule filiform, lanceolate, 5-7 mm; petiole 1-1.5 cm, hairy; leaf blade ovate or oblong, 8-18 x 4-8 cm, thinly leathery, very sparsely stellate at first and glabrescent both abaxially and adaxially, basal veins 3, laterals more than 1/2 as long as leaf blade, base rounded, margin finely crenate, apex acuminate. Panicles terminal, 4-10 cm, stellate. Bracts lanceolate. Pedicel short. Sepals oblong, 5-7 cm, hairy abaxially. Inflorescence. Terminal or axillary, cymose paniculate. Flower anatomy. Petals oblong, 3-4 mm, hairy in proximal 1/2; glands ca. 2 mm. Stamens shorter than sepals. Ovary globose, glabrous. Fruit anatomy. Drupe nearly globose or obovoid ca. 1 cm; stipe short.
31
Figure 2.1 Illustration of Microcos paniculata L.: 1, flowering branch; 2, flower; 3, lateral and adaxial view of petal; 4, infructescence (Deng, 1989).
32
Figure 2.2 Photograph of Microcos paniculata L. collected from the Kego Nature Reserve, Hatinh Province, Vietnam, in December 2008. (Taken by Dr. D. D. Soejarto, University of Illinois at Chicago)
4. Overview of phytochemical studies on the genus Microcos
Previous phytochemical work on plants in the genus Microcos has led to reports of flavonoids and other phenolic substances, piperidine alkaloids, triterpenoids, and
volatile oil components. The most rigorous structural elucidation studies have been
performed on the isolated piperidine alkaloids. The primary species of Microcos
investigated for piperidine alkaloids have been M. paniculata L. and M. philippinensis
Burret, where 2,3,6-trisubstituted piperidines containing a variety of substitution patterns
have been reported. M. paniculata produces these alkaloids to defend against herbivory,
with Bandara and associates having reported the insecticidal activity of a stem bark 33
extract of this species against the second instar larvae of the mosquito Aedes aegypti
(Bandara et al., 2000). In addition, piperidine alkaloids of this type have also been found
outside the plant kingdom, such as corydendramines A and B from the marine hydroid
Corydendrium parasiticum and the solenopsins from certain species of fire ants
(Solenopsis spp.) (Yi et al., 2003; Chen et al., 2009; Lindquist et al., 2010).
4.1 Flavonoids and other Phenolic Derivatives
Flavonoids isolated from Microcos are common throughout the plant kingdom,
and were identified from Microcos paniculata by HPLC fingerprinting employing a
calibration standard. Reported flavonoids from this species include kaempferol, vitexin,
isorhamnetin, quercetin, (-)-epicatechin, and the glycosides isorhamnetin-3-O-rutinoside,
and nodifloretin-7-O-rhamnosylglucoside (Luo et al., 1993; Fan et al., 2010; Li et al.,
2011). The trivial names, plant sources, and references for flavonoids and other phenolic
substances isolated from Microcos paniculata are summarized in Table 2.1 and Figure
2.3.
Code Trivial name Source Reference 28 Kaempferol M. paniculata Zeng et al., 2010 29 Isorhamnetin M. paniculata Luo et al., 1993 30 Isorhamnetin-3-O-rutinoside M. paniculata Li et al., 2011 31 Vitexin M. paniculata Li et al., 2011 32 Nodifloretin-7-rhamnosylglucoside M. paniculata Luo et al., 1993 33 Quercetin M. paniculata Luo et al., 1993 34 (-)-Epicatechin M. paniculata Zhang et al., 2012 35 Chlorogenic acid M. paniculata Hu and Bi, 2010 36 2-Methoxy-4-vinylphenol M. paniculata Bi et al., 2007 Table 2.1 Flavonoids and other phenolics isolated from Microcos paniculata.
34
Figure 2.3 Structures of flavonoids and other phenolics isolated from Microcos paniculata.
35
4.2 Alkaloids
Alkaloids isolated from the genus Microcos have been obtained from M. paniculata and M. philippinensis. One of the first reports was the isolation of micropine
(37) from M. philippinensis (Aguinaldo and Read, 1990). A full NMR spectroscopic dataset of 37 was reported, including nuclear Overhauser enhancements, which facilitated elucidation of 37 as (2S,3R,6S)-6-[(1E,3E,5E)-deca-1,3,5-trien-1-yl)-2-(hydroxymethyl)]-
1-methylpiperidin-3-ol. Reports of alkaloids from M. paniculata, in particular, microcosamines A and B (38, 39) and micropiperidines A-D (40-43), indicate those compounds to have a variety of functionalities around their respective piperidine rings
(Feng et al., 2008a; Luo et al., 2009). Piperidine alkaloids of this type have also been isolated from the jellyfish, Corydendrium parasiticum (corydendramines A and B; 44,
45), the sea sponge Neopetrosia proxima (neoptrosiamine A; 46), and certain fire ants in the genus Solenopsis (solenopsins A-C; 47-49) (Lindquist et al., 2000; Yi et al., 2003;
Chen et al., 2009). Other plant sources of piperidine alkaloids include the genera Azima
(Rall et al., 1967), Bathiorhamnus (Bruneton and Cavé, 1975), Conium (Roberts and
Brown, 1981), Lobelia (Ma et al., 2008), and Sedum (Colau and Hootele, 1983).
Cryptophorine (50), isolated from Bathiorhamnus cryptophorus, by Bruneton and Cavé is one of the earliest reported 2,3,6-trisubstituted piperidine alkaloids, but the substituent configuration around the piperidine ring was not determined unambiguously (Bruneton and Cavé, 1975). The trivial names, plant sources, and references for alkaloids isolated from the genus Microcos and representative examples from other natural sources are
36 summarized in Table 2.2, and their structures are presented in Figure 2.3 following the table.
Code Trivial name Source Reference 37 micropine M. philippinensis Aguinaldo and Read, 1990 38 microcosamine A M. paniculata Feng et al., 2008a 39 microcosamine B M. paniculata Feng et al., 2008a 40 micropiperidine A M. paniculata Luo et al., 2009 41 micropiperidine B M. paniculata Luo et al., 2009 42 micropiperidine C M. paniculata Luo et al., 2009 43 micropiperidine D M. paniculata Luo et al., 2009 44 corydendramine A Corydendrium Lindquist et al., 2000 parasiticum 45 corydendramine B Corydendrium Lindquist et al., 2000 parasiticum 46 neopetrosiamine A Neopetrosia proxima Wei et al., 2010 47 solenopsin A Solenopsis spp. Yi et al., 2003; Chen et al., 2009 48 solenopsin B Solenopsis spp. Yi et al., 2003; Chen et al., 2009 49 solenopsin C Solenopsis spp. Yi et al., 2003; Chen et al., 2009 50 cryptophorinine Bathiorhamnus Bruneton and Cavé, 1975 cryptophorus
Table 2.2 Piperidine alkaloids isolated from the genus Microcos and representative piperidine derivatives from other plant and animal taxa.
37
Micropine (37) Microcosamine A (38) Microcosamine B (39)
Micropiperidine A (40) Micropiperidine B (41) Micropiperidine C (42)
Micropiperidine D (43) Corydendramine A (44) Corydendramine B (45)
Neopetrosiamine A (46) Solenopsin A (47) Solenopsin B (48)
Solenopsin C (49) Cryptophorinine (50) N-Methyl-6 -(deca)- 1 ,3 ,5 -trienyl)-3 -
methoxy-2 - methylpiperidine (51)
Figure 2.4 Structures of piperidine alkaloids isolated from Microcos spp. and representative piperidine derivatives from other plant and animal taxa.
38
4.3. Triterpenoids
Triterpenoids isolated from Microcos have been elucidated structurally, and by
spectroscopic data interpretation. Specifically, friedelin, (52), maslinic acid (53), 3-trans-
feruloyl-maslinic acid (54) and 3 -O-p-hydroxy-E-cinnamoyloxy-2 ,23-dihydroxyolean-
12-en-28-oate (55), have been reported (Feng et al., 2008b; Fan et al., 2010). The trivial
names, plant sources, and references for triterpenoid components isolated from the genus
Microcos are summarized in Table 2.3, and their structures are presented in Figure 2.4
following the table.
Code Trivial name Source Reference 52 friedelin M. paniculata Feng et al., 2008b 53 maslinic acid M. paniculata Fan et al., 2010 54 3-trans-feruloyl-maslinic acid M. paniculata Fan et al., 2010 55 3 -O-p-hydroxy-E-cinnamoyloxy-2 ,23- M. paniculata Fan et al., 2010 dihydroxyolean-12-en-28-oate Table 2.3 Triterpenoids isolated from the genus Microcos.
39
3-trans-Feruloyl- Friedelin (52) Maslinic acid (53) maslinic acid (54)
3 -O-p-Hydroxy-E-cinnamoyloxy-2 ,23- dihydroxyolean-12-en-28-oate (55)
Figure 2.5 Structures of triterpenoids isolated from genus Microcos.
4.5 Biological activities of compounds reported from the genus Microcos
The free-radical-scavenging activities of 3-trans-feruloyl-maslinic acid (54), 3 -
O-p-hydroxy-E-cinnamoyloxy-2 ,23-dihydroxyolean-12-en-28-oate (55), epicatechin, and sucrose, isolated from Microcos paniculata were evaluated using the 1,1-diphenyl-2- picrylhydrazyl (DPPH), 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and
Co (II) EDTA-induced luminol chemiluminescence by flow injection in vitro bioassays
(Fan et al., 2010). Epicatechin was found to have IC50 values of 2.83 ± 0.12 µM, 1.38 ±
0.05 µM, and 0.18 ± 0.01 µM in the DPPH, ABTS and Co (II) EDTA 40
chemiluminescence assays, respectively, when compared to the ascorbic acid positive
control (Fan et al., 2010).
Insecticidal activity against the mosquito species Aedes aegypti second instar
larvae has been reported for N-methyl-6 -(deca-1 ,3 ,5 -trienyl)-3 -methoxy-2 -
methylpiperidine (51). The concentration at which 50% of larvae were moribund (MC50) and the LC50 of compound 51 were shown to be 1.0 ppm and 2.1 ppm, respectively, at 24
hours against A. aegypti second instar larvae (Bandara et al., 2000). This report suggests
that M. paniculata produces piperidine alkaloids like compound 51 to defend against
feeding by herbivory. The bis-piperidine alklaloid, neopetrosiamine A (46), was shown to
have in vitro inhibitory activity against Mycobacterium tuberculosis and Plasmodium
falciparum (Wei et al., 2010).
41
Chapter 3: Phytochemical and Bioactivity Studies on the Stem Bark, Branches, and
Leaves of Microcos paniculata
A. Statement of Problem
Literature reports on M. paniculata have demonstrated that piperidine alkaloids occur in the leaves or stem bark of the plant (Bandara et al., 2000; Feng et al., 2008; Luo et al., 2009). Since separate samples of the stem bark, branches, and leaves of M. paniculata were collected, a LC-MS dereplication procedure was developed and used to show that all three parts of the plant produce these alkaloids. Preliminary structure- activity relationship (SAR) studies to investigate the role of functional group contributions to HT-29 human colon cancer cell cytotoxicity and nicotinic acetylcholine receptor (nAChR) antagonism were carried out by undergoing isolation chemistry work and a total synthesis of the piperidinol ring. Cytotoxic and nAChR activities of piperidine alkaloids isolated from M. paniculata have not been investigated previously.
B. Experimental
1. General experimental procedures
Melting points were determined on a Fisher Scientific melting point apparatus and are uncorrected. Optical rotations were determined on a PerkinElmer model 343 automatic polarimeter (sodium lamp). UV spectra were obtained with a PerkinElmer
42
Lambda 10 UV/vis spectrometer. IR spectra were measured on a Thermo Scientific
Nicolet 6700 FT-IR spectrometer. Circular dichroism (CD) spectra were recorded on a
JASCO J-810 spectropolarimeter. Nuclear magnetic resonance (NMR) spectra were taken at room temperature on Bruker Avance DRX-300, 400 and 600 MHz NMR spectrometers, and the raw data were processed using MestReNova 6.0 software
(MestreLab Research, Santiago de Compostela, Spain). High-resolution mass spectra were recorded on a Micromass LCT ESI spectrometer, and a Micromass ESI-Tof II mass spectrometer (Micromass, Wythenshawe, UK) equipped with an orthogonal electrospray source (Z-spray) operated in the positive-ion mode. Sodium iodide was used for mass calibration for a calibration range of m/z 100-2000. Silica gel (43-60 mesh; Sorbent
Technologies, Atlanta, GA) was used for column chromatography. Dereplication was performed on a Waters Alliance 2690 Separations Module (Waters, Milford, MA) using a Waters X-Bridge C18 5 µm (4.6x100 mm) analytical column. The mobile phase flow rate was maintained at 0.75 mL/min and was split post column using a microsplitter valve
(Upchurch Scientific, Oak Harbor, WA) to ~ 20 µL/min for the introduction to the ESI source. The separations module was coupled to a Bruker amaZon speed ETD ion trap mass spectrometer with an orthogonal electrospray source operated in the positive ion mode. Scans were conducted in a range of m/z 100-1000. Analytical TLC was performed on precoated 250 µm-thickness silica gel plates (UV254, glass backed; Sorbent
Technologies). Dragendoff’s reagent (Solution A: 0.85 g bismuth nitrate in 10 mL acetic acid and 40 mL water; Solution B: 8 g potassium iodide in 20 mL water; Solutions A and
B were mixed in equal parts, and developed TLC plates were dipped once in the solution.
43
A positive reaction (indicated by a bright orange spot) was used to detect the presence of alkaloids and ethanolic sulfuric acid spray reagent (5% concentrated H2SO4 in ethanol) was used for visualization of non-UV absorbing compounds. High-performance liquid chromatography (HPLC) was conducted using a Hitachi LaChrom Elite system comprised of a L-2450 diode array detector, a L-2200 autosampler, and an L-2130 pump.
Waters X-Bridge C18 5 µm (4.6 x 150 mm) analytical and 5 µm (10 x 150 mm) ODS semi-preparative HPLC columns were used.
2. Plant material
Separate samples of the stem bark, branches and leaves of Microcos paniculata L.
(Malvaceae) were collected at the Kego Nature Reserve (18o 08.087 N; 105o 56.020 E;
40 m altitude), Hatinh Province, Vietnam, by Tran Ngoc Ninh., Vuong Tan Tu, and Djaja
Djendoel Soejarto in December 2008, who also identified this species. Microcos paniculata samples were collected under the terms of an agreement between the
University of Illinois at Chicago and the Institute of Ecology and Biological Resources
(IEBR) of the Vietnam Academy of Science and Technology, Hanoi, Vietnam. Collection of the plant material was facilitated by the Director of the Kego Nature Reserve who granted permission, and Dr. Le Xuan Canh, Director of IEBR. Voucher specimens
(numbers DDS14326 and DDS14261) documenting these plant collections have been deposited in the John G. Searle Herbarium of the Field Museum of Natural History,
Chicago, Illinois.
44
3. Extraction of the stem bark, branches, and leaves of Microcos paniculata
3.1. Extraction of M. paniculata stem bark
The dried and milled stem bark (646 g) of M. paniculata (DDS14261; program project code, A6319) was extracted with 3 L methanol four times in an 8 L conical glass percolator fitted with a glass stop-cock. The combined methanol percolate was concentrated in vacuo to form a dried red viscous methanol extract (179 g) (D1), and dissolved with 500 mL 9:1 methanol-water. The 9:1 methanol-water solution (500 mL) was defatted by partitioning with 500 mL hexane. The resulting hexane extract (D2) was obtained as a green paste (2 g). The methanol-water layer was evaporated to a thick paste, diluted with 100 mL water, and then partitioned with 500 mL of chloroform. The aqueous layer from this partition afforded 100 g of a water-soluble extract (D4), in the form of a thick, red syrup. The chloroform layer was partially detannified with 1% NaCl in water to give a chloroform-soluble extract (D3) (8 g). The aqueous layer was partitioned with ethyl acetate to afford 17 g of a viscous ethyl acetate soluble extract (D5). The fractionation scheme employed is depicted in Figure 3.1.
45
Figure 3.1 Fractionation tree employed for Microcos paniculata stem bark. (Scheme adapted from Wall et al., 1996).
3.2. Extraction of M. paniculata branches
Separate specimens of the branches of M. paniculata were collected at the Kego
Nature Reserve (18o 08.087 N; 105o 56.020 E; 40 m altitude), Hatinh Province,
Vietnam, by Tran Ngoc Ninh, Vuong Tan Tu, and Djaja Djendoel Soejarto, who also identified this species. These specimens were DDS14261 (A6320, 1.1 kg, collected in
2008) and DDS14326 (A5918, 1.2 kg, collected in 2004, and recollected in 2008 as
AA5918).
An initial batch (1.1 kg) of the dried and milled branches of M. paniculata
(DDS14261; A6320) was extracted with 6 L methanol four times in an 8 L conical glass percolator fitted with a glass stop-cock. The combined methanol percolate was concentrated in vacuo to form a dark-brown methanol extract (39 g) (D1), and dissolved 46 with 800 mL 9:1 methanol-water. The 9:1 methanol-water solution (500 mL) was defatted by partitioning with 800 mL hexane. The resulting hexane extract (D2) was obtained in the form of a green paste (7 g). The methanol-water layer was evaporated to a thick paste, diluted with 300 mL water, and then partitioned with 800 mL of chloroform.
The aqueous layer from this partition afforded approximately 15 g of a water-soluble extract (D4), as a thick brown syrup. The chloroform layer was partially detannified with
1% NaCl in water to give a chloroform-soluble extract (D3) (6 g). The fractionation scheme used for the extraction of the branches (A6320) is depicted in Figure 3.2.
A second sample (1.2 kg) of the dried and milled branches of M. paniculata
(DDS14326; A5918) was extracted with 4 L methanol three times in an 8 L conical glass percolator fitted with a glass stop-cock. The combined methanol percolate was concentrated in vacuo to form a dark-brown methanol extract (222 g) (D1), and dissolved with 700 mL 9:1 methanol-water. The 9:1 methanol-water solution (500 mL) was defatted by partitioning with 700 mL hexane. The resulting hexane extract (D2) was obtained as a green paste (34 g). The methanol-water layer was evaporated to a thick paste, diluted with 200 mL water, and then partitioned with 600 mL of chloroform. The aqueous layer from this partition afforded approximately 50 g of a water-soluble extract
(D4), as a thick brown syrup. The chloroform layer was partially detannified with 1%
NaCl in water to give a chloroform-soluble extract (D3) (10 g). The fractionation scheme used for the extraction of the second batch of branches (A5918) is depicted in Figure 3.2.
47
Plant Material (1.1 kg, dry weight A6320) (1.2 kg, dry weight A5918)
Extract with MeOH MeOH Extract (39 g, A6320) (222 g, A5918) Defat with n-hexanes
Hexane Extract Methanol-Water (9:1) (7 g, A6320) (34 g, A5918) Dilute with H2O Partition with CHCl3
CHCl3 Extract Aqueous Extract (~15 g, A6320) Wash with 1% NaCl Extract "Detannified" CHCl3 Extract (6 g, A6320) A5918 with (10 g, A5918) EtOAc
EtOAc Extract Aqueous Extract (44 g, A5918) (~50 g, A5818) (Stored)
Figure 3.2 Fractionation tree employed for Microcos paniculata branches. (Scheme adapted from Wall et al., 1996)
3.3. Extraction of M. paniculata leaves
The dried and milled leaves (770 g) of M. paniculata collected in 2008 were extracted with 3 L methanol four times in an 8 L conical glass percolator fitted with a glass stop-cock. The combined methanol percolate was concentrated in vacuo to form a dark-green methanol extract (200 g) (D1), and was dissolved with 500 mL 9:1 methanol- water. The 9:1 methanol-water solution (500 mL) was defatted by partitioning with 500 mL hexane. The resulting hexane extract (D2) was a green paste (21 g). The methanol- water layer was evaporated to a thick paste, diluted with 200 mL water, and then partitioned with 500 mL of chloroform. The aqueous layer from this partition afforded approximately 138 g of a water-soluble extract (D4), in the form of a thick green syrup. 48
The chloroform layer was partially detannified with 1% NaCl in water to give a green colored chloroform-soluble extract (D3) (12 g). The fractionation tree utilized for the leaves is depicted in Figure 3.3.
Figure 3.3. Fractionation tree employed for Microcos paniculata leaves. (Scheme adapted from Wall et al., 1996)
4. Chromatography of the chloroform-soluble extracts of the stem bark, branches and leaves of Microcos paniculata
4.1. Chromatographic purification of the stem bark of Microcos paniculata
Bioactivity-guided fractionation of the stem bark chloroform extract (IC50 = 9.9
µg/mL) was carried out using the HT-29 human colon cancer cell line to monitor cytotoxicity. Fractionation of the chloroform extract of M. paniculata stem bark (D3) (8 g), using vacuum-liquid chromatography (VLC) with 240 g silica gel (230-400 mesh),
49 employed a step gradient from hexanes, to ethyl acetate, to methanol, producing 14 aliquots. These aliquots were combined on the basis of TLC analysis to afford 11 fractions (F1-F11). The step gradient was as follows: 3 L hexanes, 1 L 25% ethyl acetate in hexanes, 2 L equal parts hexanes and ethyl acetate, 2 L 25% hexanes in ethyl acetate, 1
L ethyl acetate, 3 L 10% methanol in ethyl acetate, 1 L 15% methanol in ethyl acetate, 1
L 20% methanol and ethyl acetate, 1 L 50% methanol in ethyl acetate, and 2 L methanol wash. All collections were in 1 L increments. Fraction F6 (300 mg), eluted with 10% methanol in ethyl acetate, was selected for further purification on the basis of its in vitro cytotoxicity against the HT-29 human colon cancer cell line. Fraction 6 was visualized with Dragendorff's reagent, indicating the presence of alkaloids. Column chromatographic purification of F6 (300 mg) over 15 g of silica gel (40-63 mesh), used an isocratic mobile phase of chloroform-methanol (15:1) and provided 18 further subfractions (R1-R18). Collections were in 150 mL increments. Fraction R7 (14.1 mg) showed the presence of alkaloids as indicated using Dragendorff’s spray reagent. On charring with sulfuric acid, thin-layer chromatography using 10:1 chloroform-methanol as eluent showed pink bands around the major alkaloid compound. This was consistent with the co-elution of the alkaloids with sterols, in that the latter can turn pink after sulfuric acid treatment and heating (Sherma, 2003). Passage over Sephadex LH-20 (6 g) was used to remove these pigments from fraction R7 by elution with 100% methanol at a flow rate of approximately 1 drop/5 sec, to afford on solvent evaporation colorless needles of microgrewiapine A (fractionation code: A6319D3F6R7K1, 56; 7 mg).
50
For the fractionation of the M. paniculata stem bark ethyl acetate extract (D5) (17 g), polyamide 6 resin (Fluka, St. Louis, MO) was used. The resin (9 g) was first allowed to swell in deionized water overnight, and then two column volumes of water were eluted to equilibrate the resin. Two grams of the viscous ethyl acetate extract were subjected to sonication in 100 mL methanol for the purpose of dissolution, and applied to the top of the resin bed. A step gradient in 300 mL fraction collections was used consisting of the following: 1 L of pure deionized water, 1 L water with 10% methanol, 1 L water with
40% methanol, 1 L methanol with 10% water, and finally a column wash with 1 L aqueous 1% NaCl, to produce 16 aliquots. These aliquots were combined on the basis of
TLC analysis into four fractions (F1-F4). Fraction F2 (3.5 mg), eluted with water and
10% methanol, produced a white residue when evaporated to dryness, which was identified as palmitic acid (fractionation code: A6319D6F2K1, 57; 3.5 mg).
4.2. Chromatographic purification of the branches of Microcos paniculata
Chloroform extracts from specimens of the branches (A6320 and A5918) were chromatographed separately. Bioactivity-guided fractionation of the chloroform extract from A6320 (IC50 = 13.1 µg/mL) was carried out using the HT-29 human colon cancer cell line. Fractionation of the A6320 chloroform extract (D3) (6 g) over 180 g silica gel
(230-400 mesh), used a step gradient from hexanes, to ethyl acetate, to methanol, with the fractions obtained pooled into nine major fractions. The step gradient was as follows: 1 L hexanes, 1 L 5% ethyl acetate in hexanes, 1.2 L 10% ethyl acetate in hexanes, 4 L 15% ethyl acetate in hexanes, 4 L 20% ethyl acetate in hexane, 4 L 30% ethyl acetate in
51 hexane, 6 L equal parts hexane and ethyl acetate, 1 L 60%, 70% and pure ethyl acetate respectively, 2 L 10% methanol in ethyl acetate, 1 L 20% methanol in ethyl acetate, 1 L
30% methanol in ethyl acetate, 40% methanol in ethyl acetate, 50% methanol in ethyl acetate, and 1 L methanol wash. All collections were in 350 mL increments, and thin- layer chromatography was used to combine like fractions. This produced nine pooled fractions (F1-F9) based on TLC analysis of the column aliquots, with the most pronounced cytotoxicity found for F7 (330 mg) (IC50 = 11.6 µg/mL) and F8 (1.55 g) (IC50
= 14.3 µg/mL), eluted using EtOAc with 10% MeOH. A positive response to
Dragendorff’s reagent suggested that one or more alkaloids in F7 and F8 could be contributing to the observed cytotoxicity. Purification of F7 (330 mg) with a reversed- phase (C18) HPLC gradient separation, starting at 30:70 CH3CN-H2O, and increasing to
60:40 CH3CN-H2O over 40 min, afforded 7 -(3 ,4 -dihydroxyphenyl)-N-[4- methoxyphenyl)ethyl]propenamide (fractionation code: A6320D3F7K1, 58; 3 mg) (tR =
22 min) as the most highly absorbing analyte in the mixture at 205 nm. Compound 58 was isolated as a white semi-crystalline residue.
Purification of F8 (1.5 g) over 30 g of silica gel (43-60 mesh), used an isocratic mobile phase consisting of 15:1 chloroform-methanol. The eluent was collected in 100 mL increments, resulting in 34 aliquots that were combined into two fractions (R1 and
R2) based on TLC analysis. Fraction R1 (8.1 mg) yielded a yellow crystalline material that was further purified by reversed-phase HPLC (C18), using a water to methanol gradient (5-95% over 60 min), with liriodenine (fractionation code: A6320D3F8R1K1,
59; 1.3 mg) eluting at tR = 30 min. Fraction R2 (544.2 mg) was purified by passage over a 52
column containing silica gel (15 g) (40-63 µm mesh) eluted with 10:1:0.1 chloroform-
methanol-acetic acid), in 20 mL increments, producing 30 aliquots. These aliquots were
combined based on TLC analysis into four fractions (T1-T4). Fractions T2 (28.5 mg) and
T3 (30 mg) showed a group of closely eluting alkaloids that were only partially resolved
under normal-phase TLC conditions (Rf = 0.2 for the major alkaloid zone of fraction T2;
Rf = 0.4 for the major alkaloid zone of fraction T3; 5:1:0.1 chloroform-methanol-acetic
acid). Reversed-phase HPLC (C18) separation of fraction T2 using 48:52 MeOH-H2O
with 0.01% NH4OH in water as mobile phase, afforded microgrewiapine B (fractionation
code: A6320D3F8R2T2K2, 60; 2 mg) (tR = 87 min) as the most highly UV absorbing
analyte in the mixture at 270 nm. Reversed-phase HPLC (C18) separation of fraction T3
using 30:70 CH3CN-H2O with 0.01% NH4OH in water mobile phase, afforded
microgrewiapine C (fractionation code: A6320D3F8R2T3K3, 61; 1.5 mg) (tR = 79 min)
as the most highly UV absorbing analyte in the mixture at 270 nm.
Fraction F6 (126 mg), which eluted with pure ethyl acetate from the initial silica
gel column of the chloroform extract, was further purified on the basis of TLC analysis
suggesting a terpenoid compound, since it gave a pink color after spraying with H2SO4 and heating. Fraction F6 was subjected to HPLC analysis using a gradient elution with methanol to water (5% to 95% over 40 min), to afford maslinic acid (fractionation code:
A6320D3F6K1, 62; 1 mg) (tR = 48 min) as a white powder.
Bioactivity-guided fractionation of the chloroform extract from A5918 (IC50 =
10.1 µg/mL) was carried out using the HT-29 human colon cancer cell line. Fractionation
53 of the A5918 chloroform extract (10 g) over 270 g silica gel (230-400 mesh), used a step gradient from hexanes, to ethyl acetate, to methanol, with the 81 aliquots obtained combined into six fractions (F1-F6) on the basis of TLC analysis. The step gradient was as follows: 3 L hexanes, 4 L 10% ethyl acetate in hexanes, 4 L 20% ethyl acetate in hexane, 4 L 30% ethyl acetate in hexane, 2 L 40% ethyl acetate in hexane, 2 L equal parts hexane and ethyl acetate 50:50, 2 L pure ethyl acetate with 25% hexane, 2 L 10% methanol in ethyl acetate, 1 L 30% methanol in ethyl acetate, and 1 L methanol wash. All collections were in 300 mL increments. The most pronounced cytotoxicity was observed for fractions F3 (IC50 = 13.3 µg/mL) and F4 (IC50 = 11.7 µg/mL), eluted using hexane-
30% EtOAc to equal parts hexane and ethyl acetate. It was noted that F3 and F4 exhibited approximately the same cytotoxic potency as the crude chloroform extract. Fraction F4
(341 mg) was subjected to passage over Sephadex LH-20 (5 g), and eluted isocratically with pure MeOH in 50 mL collections, affording ten pooled fractions (R1-R10). Fraction
R8 produced a white residue as the methanol evaporated, to afford daucosterol
(fractionation code: A5918D3F4R8K1, 63, 15 mg).
Bioactivity-guided fractionation of the ethyl acetate extract from A5918 (D6)
(IC50 = 16.8 µg/mL) was carried out using the HT-29 human colon cancer cell line.
Fractionation of the ethyl acetate extract (44 g) was conducted over 500 g silica gel (230-
400 mesh), using an isocratic mobile phase (chloroform-methanol, 20:1), with the 47 aliquots obtained combined into eight fractions (F1-F8) on the basis of TLC analysis. All collections were in 300 mL increments. The most pronounced cytotoxicity was for fraction F8 (1.2 g) (IC50 = 14.9 µg/mL), although this fraction had essentially the same 54 cytotoxicity as the crude ethyl acetate extract. Fraction F8 (101.9 mg) was purified by preparative TLC (20 X 20 cm, 500 µm thickness), developed by dichloromethane- acetone (3:1), to yield (-)-epicatechin (fractionation code: A5918D6F8K2, 34; 90 mg).
4.3. Chromatographic purification of the leaves of Microcos paniculata
Bioactivity-guided fractionation of the chloroform extract from the leaf sample
A6317 (D3) (IC50 = 2.9 µg/mL) was carried out using the HT-29 human colon cancer cell line. Fractionation of the leaf chloroform extract (12 g) over 300 g silica gel (230-400 mesh), used a step gradient from hexanes, to ethyl acetate, to methanol, with the fractions obtained pooled into eleven major fractions (F1-F11). The step gradient was as follows: 3
L hexanes, 4 L 10% ethyl acetate in hexanes, 4 L 20% ethyl acetate in hexane, 8 L 30% ethyl acetate in hexane, 2 L 40% ethyl acetate in hexane, 2 L equal parts hexane and ethyl acetate 50:50, 2 L pure ethyl acetate with 25% hexane, 2 L 10% methanol in ethyl acetate, 2 L 30% methanol in ethyl acetate, and 1 L methanol wash. All collections were in 1 L increments. The most pronounced cytotoxicity was found for fraction F11 (IC50 =
1.7 µg/mL); HT-29 cells, separated using ethyl acetate-40% MeOH. Fraction F11 (2.03 g) was loaded onto a Diaion HP-20 column (52 g), and eluted isocratically with 50:50 methanol-water with approximately 2 L mobile phase, until a red-orange residue (R1, 400 mg), corresponding to a Dragendorff-positive alkaloidal fraction, was washed out. The retained chlorophyll portion (R2) (390.9 g) was eluted with pure acetone, and stored.
Fraction R was subjected to silica gel (43-63 mesh) chromatography using a gradient mobile phase of 70:10:5:5 ethyl acetate-acetonitrile-methanol-water, increasing to
55
60:20:20:20 of this same solvent mixture (50 mL collections). This produced 50 aliquots that were combined on the basis of TLC analysis into four fractions (T1-T4). Fraction T1
(168.2 mg) was further separated by reversed-phase HPLC (C18) (56:44 acetonitrile-H2O with 0.01% NH4OH in water mobile phase), to afford crude microcosamine A (tR = 46 min, 4.5 mg). This crude alkaloid was subjected to silica gel (5 g) (43-63 mesh) chromatography (chloroform-methanol, 8:1) (1 mL collections), producing 21 aliquots that were combined on the basis of TLC analysis into two fractions (H1-H2). From fraction H2 (2.0 mg) was afforded microcosamine A (fractionation code:
A6317D3F11R1T1H2K1, 64; 0.6 mg) as an amorphous solid.
Fraction F6 (293 mg) (IC50 = 10.1 µg/mL) of sample A6317, eluted with hexane-
30% ethyl acetate from the initial chloroform extract (D3), was further purified isocratically using 15 g silica gel (43-63 mesh) (hexane-ethyl acetate, 3:1, 25 mL collections). The 96 aliquots obtained were combined on the basis of TLC analysis into five fractions (R1-R5). Fraction R4 (54.1 mg), was subjected to reversed-phase (C18)
HPLC (5:95 methanol-water increasing to 95:5 methanol-water over 40 min), to afford
(-)-loliolide (fractionation code: A6317D3F6R4K1, 65; 3.6 mg) (tR = 20 min), at a detection wavelength of 220 nm, as a white powder. The structures of the compounds isolated from Microcos paniculata stem bark (A6319), branches (A6320, A5918), and leaves (A6317) in the present study are shown in Figure 3.4.
56
Microgrewiapine A (56) Microgrewiapine B (60)
7 -(3 ,4 -Dihydroxyphenyl)-N- Microgrewiapine C (61) [4- methoxyphenyl)-ethyl]propenamide (58)
Liriodenine (59) Maslinic acid (62) (-)-Loliolide (65)
Daucosterol (63) Microcosamine A (64)
(-)-Epicatechin (34) Palmitic acid (57)
Figure 3.4 Summary of compounds isolated from Microcos paniculata stem bark, branches, and leaves in the present investigation. 57
5. Dereplication of the stem bark, branches, and leaves of M. paniculata
5.1. Occurrence of piperidine alkaloids in different Microcos plant parts
Dereplication has been formally defined as the attempt to determine the occurrence of previously known active compounds in crude extracts as early as possible to minimize the effort lost in their isolation (Constant and Beecher, 1995; Dinan, 2006).
In a practical sense, dereplication procedures are carried out typically on extracts of newly investigated plant species to prevent re-isolating known compounds that have been found in other plant species. However, dereplication may also be applied to extracts from different parts of the same plant. This thesis work has investigated compounds from the stem bark, branches and leaves of M. paniculata. However, before isolation work began, it was not known if all these plant parts produce piperidine alkaloids. The majority of earlier reports on the isolation of piperidine alkaloids from Microcos spp. were performed using samples of the leaves (Aguinaldo and Read, 1990; Feng et al., 2008a; Luo et al.,
2009) or stem bark (Bandara et al., 2000). A dereplication method was accomplished in this work, to ascertain if the branches of M. paniculata also produce piperidine alkaloids, so as not to commit time to the fractionation of this plant part if such compounds were absent.
5.2. Dereplication procedure
Separate chloroform extracts of the stem bark, branches, and leaves of M. paniculata were prepared. The extracts were made at 20 µg/mL and 5 µg/mL concentrations and injected into a Hitachi LaChrom Elite system equipped with a L-2450
58 diode array detector, a L-2200 autosampler, and a L-2130 pump. The separation was carried out using a Waters X-Bridge C18 5 µm (4.6x100 mm) analytical column with a gradient separation consisting of 0-17 min (60:40 methanol-water), 18-19 min (ramp to
95:5 methanol-water), 20-30 min (ramp to 100:0 methanol-water), at a flow rate of 0.75 mL/min. The aqueous mobile phase contained 0.005% NH4OH with the eluent from the
HPLC separation collected directly into a 96-well plate at 20 sec intervals (250 µL/well)
(slots A1 to H6 for HT-29 human colon cancer cell line cytotoxicity testing). Wells H-7 to H-12 were left blank for a DMSO negative control. Cytotoxicity results were expressed as percent cell survival, as shown in Table 3.1. Chloroform extracts of the stem bark (SB), branches (BR) and leaves (LF) were dissolved in methanol and diluted to a concentration of 5 µg/mL before LC-MS experiments. The LC-MS conditions for dereplication employed the same separation conditions for as used for cytotoxicity evaluation, on a Waters Alliance 2690 Separations Module (Waters, Milford, MA) using a Waters X-Bridge C18 5 µm (4.6 x100 mm) analytical column. The mobile phase flow rate was maintained at 0.75 mL/min and was split post column using a microsplitter valve
(Upchurch Scientific, Oak Harbor, WA) to ~20 µL/min for the introduction to the ESI source. The separations module was coupled to a Bruker amaZon speed ETD ion trap mass spectrometer with an orthogonal electrospray source operated in the positive-ion mode. Sodium iodide was used for mass calibration for a calibration range of m/z 100-
1000. Optimal ESI conditions were: capillary voltage 3000 V, source temperature 110oC and a cone voltage of 55 V. The ESI gas was nitrogen. Q1 was set to optimally pass ions from m/z 100–1000 and all ions transmitted into the pusher region of the TOF analyzer
59 were scanned over m/z 100-1000 with a 1 sec integration time. Data were acquired in continuum mode during the LC run.
5.3. Dereplication results
Percent survival values and total ion chromatograms (Tables 3.1 and 3.2) showed that the leaf and stem bark extracts contained compounds showing m/z values of 264.4, eluting at approximately 21 minutes with the most potent cytotoxicity. These most potent fractions are illustrated as shaded boxes in these tables. For all three plant parts, the most potent activity occurred when the mobile phase composition was approximately 80:20 methanol-water, which is consistent with the polarity of microgrewiapines A-C (56, 60,
61). The leaf chloroform extract was the most active, having the largest number of wells in the 96-well plate showing low cell survival. Specifically, the leaf chloroform extract showed active chemical constituents eluting at 5 µg/mL from ~23-24 min and at 20
µg/mL from ~22-25 minutes (Table 3.1). The total ion chromatograms for the stem bark, leaves, and branches exhibited peaks at m/z 264.4 and m/z 280.4, with possible molecular
+ + formulas of C17H29NO + H and C17H29NO2 + H corresponding to microgrewiapine A
(56), and microgrewipines B and C (60, 61), respectively. The mass spectrometric data showed that all three organs of M. paniculata investigated produce piperidine alkaloids with molecular formulas that correspond to the actual pure compounds isolated (56, 60,
61) (Tables 3.1-3.3).
60
Leaf extract 96-well plate at 20 µg/mL 1 2 3 4 5 6 7 8 9 10 11 12 A 1.00 0.95 0.96 0.94 0.24 0.84 0.80 0.74 0.84 0.79 0.81 0.90 B 0.94 1.03 1.12 1.13 1.13 1.07 0.96 0.95 0.92 0.98 0.92 0.94 C 1.04 1.09 1.26 1.25 1.24 1.16 1.01 1.12 1.25 0.98 0.92 0.84 D 0.97 1.01 1.19 1.23 1.23 1.23 1.07 1.11 1.18 1.05 0.96 0.85 E 1.01 1.08 1.22 1.24 1.21 1.17 1.06 1.09 1.05 1.01 0.94 0.88 F 0.97 1.05 1.19 1.16 0.58 0.07 0.08 0.09 0.10 0.10 0.10 0.55 G 0.55 0.53 0.92 0.91 0.94 0.77 0.97 0.88 0.95 0.94 0.93 0.97 H 0.95 0.94 1.00 0.99 1.00 0.96 1.01 0.98 0.97 0.90 0.89 0.95 Leaf extract 96-well plate at 5 µg/mL 1 2 3 4 5 6 7 8 9 10 11 12 A 0.89 0.93 0.96 0.86 0.85 0.89 0.84 0.89 0.86 0.90 0.93 1.03 B 1.01 1.08 1.07 1.00 1.05 0.99 1.02 1.03 0.99 1.04 1.01 0.94 C 0.88 1.05 1.08 1.08 1.02 1.05 1.01 1.02 1.03 1.07 0.95 0.98 D 1.01 1.01 1.14 1.11 1.13 1.14 1.18 1.11 1.15 1.13 1.02 0.93 E 1.03 1.03 1.09 1.12 1.12 1.13 1.10 1.30 1.11 1.15 1.04 0.87 F 0.90 1.08 1.08 1.06 0.99 0.67 0.46 0.46 0.31 0.28 0.88 0.84 G 0.75 0.90 0.99 0.94 1.01 1.08 0.96 0.99 1.04 1.03 1.00 0.92 H 1.00 1.07 0.98 1.04 1.03 1.01 0.98 0.10 0.96 0.97 1.00 0.96 Total ion chromatogram of leaf chloroform extract
Peaks at m/z 264.4 with molecular formula corresponding to microgrewiapine A (56)
Table 3.1 Dereplication of M. paniculata chloroform extract of the leaves. Grids illustrate 96- well plates at (A) 20 µg/mL, and (B) 5 µg/mL concentration of the respective chloroform extracts. Numerical values in the grid represent the percent survival of HT-29 human colon cancer cells at the specified concentration. Shaded boxes exhibited the lowest cell survival.
61
Stem bark extract 96-well plate at 20 µg/mL 1 2 3 4 5 6 7 8 9 10 11 12 A 0.66 0.63 0.66 0.65 0.69 0.63 0.59 0.60 0.60 0.71 0.76 0.67 B 0.74 0.74 0.77 0.79 0.46 0.76 0.73 0.78 0.77 0.80 0.81 0.70 C 0.74 0.79 0.87 0.87 0.92 0.90 0.86 0.86 0.90 0.85 0.84 0.75 D 0.76 0.81 0.89 0.88 0.95 0.93 0.93 0.87 0.92 0.89 0.89 0.76 E 0.73 0.84 0.88 0.91 0.95 0.94 0.91 0.91 0.93 0.87 0.93 0.82 F 0.73 0.82 0.86 0.90 0.14 0.07 0.07 0.08 0.10 0.09 0.08 0.08 G 0.07 0.10 0.27 0.22 0.20 0.17 0.33 0.32 0.21 0.66 0.52 0.74 H 0.73 0.74 0.70 0.70 0.71 0.73 0.85 0.91 0.94 0.92 0.89 0.90 Stem bark 96-well plate at 5 µg/mL 1 2 3 4 5 6 7 8 9 10 11 12 A 0.78 0.82 0.84 0.84 0.85 0.82 0.82 0.83 0.85 0.82 0.85 0.88 B 0.88 0.93 0.94 0.89 0.92 0.89 0.91 0.89 0.91 0.91 0.90 0.86 C 0.93 0.93 0.99 1.00 1.01 0.98 1.01 1.01 1.03 1.01 0.91 0.86 D 0.85 1.02 0.98 1.02 1.05 1.01 1.01 0.99 1.04 0.99 0.92 0.86 E 0.86 0.94 1.00 1.00 1.04 1.06 1.03 1.00 1.06 1.01 1.00 0.95 F 0.84 0.96 0.99 0.99 1.01 0.96 0.78 0.71 0.85 0.82 0.59 0.82 G 0.82 0.94 0.93 0.94 0.93 0.88 0.93 0.93 0.92 0.91 0.86 0.92 H 0.84 0.84 0.85 0.87 0.85 0.86 0.90 0.87 0.96 0.88 0.92 0.94 Total ion chromatogram of stem bark chloroform extract
Peaks at m/z 264.4 with molecular formula corresponding to microgrewiapine A (56)
Table 3.2 Dereplication of M. paniculata chloroform extract of the stem bark. Grids illustrate 96-well plates at (A) 20 µg/mL, and (B) 5 µg/mL concentration of the respective chloroform extracts. Numerical values in the grid represent the percent survival of HT-29 human colon cancer cells at the specified concentration. Shaded boxes exhibited the lowest cell survival.
62
Branches extract 96-well plate at 20 µg/mL 1 2 3 4 5 6 7 8 9 10 11 12 A 0.66 0.63 0.66 0.65 0.69 0.63 0.59 0.60 0.60 0.71 0.76 0.67 B 0.74 0.74 0.77 0.79 0.46 0.76 0.73 0.78 0.77 0.80 0.81 0.70 C 0.74 0.79 0.87 0.87 0.92 0.90 0.86 0.86 0.90 0.85 0.84 0.75 D 0.76 0.81 0.89 0.88 0.95 0.93 0.93 0.87 0.92 0.89 0.89 0.76 E 0.73 0.84 0.88 0.91 0.95 0.94 0.91 0.91 0.93 0.87 0.93 0.82 F 0.73 0.82 0.86 0.90 0.14 0.07 0.07 0.08 0.10 0.09 0.08 0.08 G 0.07 0.10 0.27 0.22 0.20 0.17 0.33 0.32 0.21 0.66 0.52 0.74 H 0.73 0.74 0.70 0.70 0.71 0.73 0.85 0.91 0.94 0.92 0.89 0.90 Branches 96-well plate at 5 µg/mL 1 2 3 4 5 6 7 8 9 10 11 12 A 0.78 0.82 0.84 0.84 0.85 0.82 0.82 0.83 0.85 0.82 0.85 0.88 B 0.88 0.93 0.94 0.89 0.92 0.89 0.91 0.89 0.91 0.91 0.90 0.86 C 0.93 0.93 0.99 1.00 1.01 0.98 1.01 1.01 1.03 1.01 0.91 0.86 D 0.85 1.02 0.98 1.02 1.05 1.01 1.01 0.99 1.04 0.99 0.92 0.86 E 0.86 0.94 1.00 1.00 1.04 1.06 1.03 1.00 1.06 1.01 1.00 0.95 F 0.84 0.96 0.99 0.99 1.01 0.96 0.78 0.71 0.85 0.82 0.59 0.82 G 0.82 0.94 0.93 0.94 0.93 0.88 0.93 0.93 0.92 0.91 0.86 0.92 H 0.84 0.84 0.85 0.87 0.85 0.86 0.90 0.87 0.96 0.88 0.92 0.94 Total ion chromatogram of branches chloroform extract
Peaks at m/z 280.4 with molecular formula corresponding to microgrewiapine B and C (60, 61)
Table 3.3 Dereplication of M. paniculata chloroform extract of the branches. Grids illustrate 96-well plates at (A) 20 µg/mL, and (B) 5 µg/mL concentration of the respective chloroform extracts. Numerical values in the grid represent the percent survival of HT-29 human colon cancer cells at the specified concentration. Shaded boxes exhibited the lowest cell survival.
63
6. Characterization of isolated compounds
6.1 Characterization of microgrewiapine A (56)
25 Microgrewiapine A (56). Colorless needle crystals; mp 127-128 °C; [ ]D +15.4
(c 0.1, MeOH); UV (MeOH) max (log ) 270 (3.87) nm; IR (film) vmax 3402, 2956, 2923,
-1 1 2864 cm ; H NMR (600 MHz, CDCl3) H 0.89 (3H, t, J = 7.2 Hz, H-10 ), 1.20 (3H, d, J
= 6.1 Hz, 2-CH3), 1.29-1.38 (4H, m, H-4 , H-8 -H-9 ), 1.49 (1H, ddd, J = 10.1, 3.0, 3.0
Hz, H-5 ), 1.63 (1H, m, H-5 ), 1.83 (1H, dq, J = 8.9, 6.1 Hz, H-2), 2.03 (1H, m, H-4 ),
2.09 (2H, dt, J = 7.2, 6.9, H-7 ), 2.21 (3H, s, N-CH3), 2.48 (1H, ddd, J = 11.2, 8.7, 3.0 Hz,
H-6), 3.27 (1H, ddd, J = 10.8, 8.9, 4.5 Hz, H-3), 5.53 (1H, dt, J = 14.6, 8.8 Hz, H-1 ),
5.71 (1H, dd, J = 14.5, 7.4, H-6 ), 6.03-6.16 (4H, m, H-2 -H-5 );13C NMR (150 MHz,
CDCl3) C 136.7 (C-1 ), 135.7 (C-6 ), 130.1-132.6 (C-2 - C-5 ), 72.9 (C-3), 67.5 (C-6),
66.1 (C-2), 40.3 (N-CH3), 33.5 (C-4), 32.5 (C-7 ), 31.4 (C-8 ), 31.2 (C-5), 22.0 (C-9 ),
+ + 16.5 (CH3-2), 14.1 (C-10 ); HRESIMS m/z 264.2329 [M+H] (calcd for C17H29NO + H ,
264.2327). The purity of microgrewiapine A (56) was verified using thin-layer chromatography (TLC) with three solvent systems (5:1 chloroform-methanol + 0.1%
AcOH, Rf = 0.5); 20:1 ethyl acetate-methanol, Rf = 0.3; 1:3 dichloromethane-acetone, Rf
= 0.4).
A catalytic amount of N,N-dimethylaminopyridine (DMAP) (0.1 equiv.) and 5 mg of 56 were placed in a round-bottomed flask under argon. Anhydrous dichloromethane
(500 L) was added, and the mixture was cooled to 0 °C. Triethylamine (2 equiv.) was added, followed by dropwise addition of acetic anhydride (3 equiv.), and the reaction was
64
warmed to room temperature and stirred for 3 h. The reaction was quenched with
saturated aqueous NaHCO3 solution at 0 °C. The mixture was extracted two times with
ethyl acetate (50 mL), and the combined organic layers were washed with brine and dried
over Na2SO4, filtered, and evaporated in vacuo. The crude product was chromatographed
over silica gel using chloroform-methanol-acetic acid, 10:1:0.1) to yield microgrewiapine
A 3-acetate as an amorphous solid (3 mg, 60%). Microgrewiapine A 3-acetate exhibited:
25 1 [ ]D +19.5 (c 0.1, MeOH); UV (MeOH) max (log ) 259 (4.29) nm; H NMR (400 MHz,
CDCl3) H 0.87 (3H, t, J = 7.0 Hz, H-10 ), 1.11 (3H, d, J = 5.8 Hz, 2 -CH3), 1.30 (1H, m,
H-2 ), 1.31-1.42 (1H, m, H-4 ), 1.31-1.42 (2H, m, H-8 -9 ), 1.35 (1H, m, H-5 ), 1.55
(1H, ddd, J = 13.2, 3.3, 2.0 Hz, H-5 ), 1.69 (1H, m, H-4 ), 2.05 (3H, s, -OCOCH3), 2.12
(1H, m, H-7 ), 2.21 (3H, s, N-CH3), 2.54 (1H, ddd, J = 11.2, 8.6, 3.4 Hz, H-6 ), 4.48 (1H,
ddd, J = 10.6, 7.3, 2.6 Hz, H-3 ), 5.55 (1H, dt, J = 14.4, 8.8 Hz, H-1 ), 5.72 (1H, dd, J =
13 7.1, 7.0 Hz, H-6 ), 6.04-6.18 (4H, m, H-2 -5 ); C NMR (100 MHz, CDCl3) C 170.7 (-
OCOCH3), 137.4 (C-1 ), 136.7 (C-6 ), 130.8-133.6 (C-2 - C-5 ), 75.1 (C-3), 67.2 (C-6),
33.7 (C-7 ), 32.3 (C-4), 31.4 (C-8 ), 30.3 (C-5), 22.4 (C-9 ), 21.8 (C-2), 14.7 (C-10 );
+ HRESIMS m/z 306.2419 [M+H] (calcd for C19H31NO2, 306.2433). The purity of
microgrewiapine A 3-acetate was verified using thin-layer chromatography (TLC) with
three solvent systems (5:1 chloroform-methanol + 0.1% acetic acid, Rf = 0.8; 20:1 ethyl
acetate-methanol, Rf = 0.6; 1:3 dichloromethane-acetone, Rf = 0.6).
6.2 Characterization of microgrewiapine B (60)
25 Microgrewiapine B (60). Colorless needle crystals; mp 134-136 °C; [ ]D +4.0
(c 0.1, MeOH); UV (MeOH) max (log ) 272 (3.97) nm; IR (film) vmax 3400, 2900, 1600 65
-1 1 cm ; H NMR (400 MHz, CDCl3) H 0.89 (3H, t, J = 7.0 Hz, H-10 ), 1.26-1.41 (4H, m,
H-8 -9 ), 1.46 (1H, m, H-4 ), 1.57 (3H, d, J = 6.0 Hz, 2-CH3), 1.62 (1H, m, H-5 ), 2.09
(2H, dt, H-7 ), 2.13 (1H, m, H-4 ), 2.33 (1H, m, 5 ), 2.86 (1H, dq, J = 10.7, 6.1 Hz, H-2),
2.95 (3H, s, N-CH3), 3.44 (1H, m, H-6), 3.90 (1H, ddd, J = 11.2, 10.5, 4.8 Hz, H-3), 5.74
(1H, dd, J = 14.2, 7.2 Hz, H-6 ), 5.94 (1H, dt, J = 15.3, 8.9 Hz, H-1 ), 6.05-6.26 (4H, m,
13 H-2 -5 ); C NMR (100 MHz, CDCl3) C 137.6 (C-6 ), 135.8-128.8 (C-5 -C-2 ), 127.7
(C-1 ), 78.8 (C-6), 76.5 (C-2), 67.4 (C-3), 51.1 (N-CH3), 33.0 (C-4), 32.9 (C-7 ), 30.9 (C-
+ 8 ), 26.6 (C-5), 21.8 (C-9 ), 14.3 (C-10 ), 10.6 (CH3-2); HRESIMS m/z 280.2269 [M+H]
(calcd for C17H29NO2, 280.2276). The purity of microgrewiapine B (60) was supported
using thin-layer chromatography (TLC) with three solvent systems (60:10:5:5 relative
volumes of ethyl acetate-acetonitrile-water-methanol, Rf = 0.3; 5:1 chloroform-methanol
+ 0.005% NH4OH, Rf = 0.65; 50:50 methanol-water, C18 TLC plate, Rf = 0.4).
6.3 Characterization of microgrewiapine C (61)
25 Microgrewiapine C (61). Colorless needle crystals; mp 130-131 °C; [ ]D +77.8
(c 0.1, MeOH); UV (MeOH) max (log ) 274 (3.92) nm; IR (film) vmax 3400, 2900, 1600
-1 1 cm ; H NMR (400 MHz, CDCl3) H 0.89 (3H, t, J = 7.1 Hz, H-10 ), 1.24-1.39 (4H, m,
H-8 -9 ), 1.57 (1H, m, H-4 ), 1.66 (3H, d, J = 6.5 Hz, 2-CH3), 2.01 (1H, m, 4 ), 2.09
(2H, dt, J = 7.0, 6.2 Hz, H-7 ), 2.67 (1H, ddd, J = 12.0, 4.1, 3.6 Hz, H-5 ), 2.86 (3H, s, N-
CH3), 3.05 (1H, dq, J = 12.7, 6.5 Hz, H-2), 3.53 (1H, ddd, J = 11.4, 9.4, 1.9 Hz, H-6),
3.82 (1H, bs, H-3), 5.72 (1H, dd, J = 15.2, 9.0 Hz, H-6 ), 5.98 (1H, dt, J = 15.1, 7.0 Hz,
13 H-1 ), 5.96-6.25 (4H, m, H-2 -5 ); C NMR (100 MHz, CDCl3) C 137.8 (C-6 ), 136.1-
129.2 (C-2 - C-5 ), 127.9 (C-1 ), 79.5 (C-6), 71.1 (C-3), 68.6 (C-2), 54.1 (N-CH3), 32.7 66
(C-7 ), 32.1 (C-4), 31.5 (C-8 ), 23.8 (C-5), 22.5 (C-9 ), 14.1 (C-10 ), 13.5 (CH3-2);
+ + HRESIMS m/z 280.2281 [M+H] (calcd for C17H29NO2 + H , 280.2277). The purity of microgrewiapine C (61) was supported using thin-layer chromatography (TLC) with three solvent systems (60:10:5:5 relative volumes of ethyl acetate-acetonitrile-water- methanol, Rf = 0.3; 5:1 chloroform-methanol + 0.005 NH4OH, Rf = 0.65; 50:50 methanol-water, C18 TLC plate, Rf = 0.4).
6.4 Characterizationof microcosamine A (64)
25 1 Microcosamine A (64). Amorphous solid; [ ]D +4 (c 0.1, MeOH); H NMR
(600 MHz, CDCl3) H 0.90 (3H, t, J = 7.2 Hz, H-10 ), 1.27-1.32 (1H, m, H-4), 1.32 (3H, m, 2-CH3), 1.34-1.39 (4H, m, H-8 -9 ), 1.55-1.65 (1H, m, H-5 ), 2.10 (2H, dt, J = 7.3, 6.9
Hz), 2.69 (1H, m, H-2), 3.36 (1H, m, H-3), 3.41 (1H, m, H-6), 5.65 (1H, dt, J = 15.1, 7.4
Hz, H-6 ), 5.74 (1H, dd, J = 14.6, 7.0 Hz, H-1 ), 6.02-6.28 (4H, m, H-2 -5 ); 13C NMR
(150 MHz, CDCl3) C 138.3 (C-1 ; C-6 ), 138.3-131.1 (C-2 -C-5 ), 74.4 (C-3), 59.9 (C-6),
59.5 (C-2), 33.6 (C-4; C-7 ), 32.4 (C-5; C-8 ), 23.9 (C-9 ), 20.1 (2-CH3), 14.9 (C-10 );
+ HRESIMS m/z 250.2163 [M+H] (calcd for C16H28NO, 250.2171).
6.5 Characterization of liriodenine (59)
Liriodenine (59). Yellow amorphous powder; UV (MeOH) max (log ) 408
-1 1 (3.16), 296 (3.47) nm; IR (film) vmax 2950, 1650, 1580, 730 cm ; H NMR (400 MHz,
CDCl3) H 6.36 (2H, s, -OCH2O-), 7.16 (1H, s, H-3), 7.56 (1H, t, J = 7.5 Hz, H-9), 7.71
(1H, t, J = 7.2 Hz, H-10), 7.75 (1H, d, J = 4.8 Hz, H-4), 8.56 (1H, d, J = 7.7 Hz, H-8),
8.60 (1H, d, J = 8.1 Hz, H-11), 8.87 (1H, d, J = 5.0 Hz, H-5); 13C NMR (100 MHz,
67
CDCl3) C 182.8 (C-7), 152.1 (C-2), 148.5 (C-2), 145.7 (C-6a), 145.3 (C-5), 136.1 (C-3a),
134.3 (C-10), 133.2 (C-11a), 131.6 (C-7a), 129.2 (C-8), 128.9 (C-9), 127.7 (C-11a),
124.7 (C-4), 123.6 (C-3b), 108.5 (C-1a), 103.6 (C-3), 102.8 (-OCH2O-); HRESIMS m/z
+ 298.0507 [M+Na] (calcd for C17H9NO3Na, 298.0480). The purity of liriodenine (59)
was supported using thin-layer chromatography (TLC) with three solvent systems (10:1
chloroform-methanol, Rf = 0.4; 20:1 ethyl acetate-methanol, Rf = 0.2; 70:30 methanol-
water, Rf = 0.3).
6.6 Characterization of 7 -(3 ,4 -dihydroxyphenyl)-N-[4-
methoxyphenyl)ethyl]propenamide (58)
7 -(3 ,4 -Dihydroxyphenyl)-N-[4-methoxyphenyl)ethyl]propenamide (58).
Colorless solid; mp 101-102 °C; UV (MeOH) max (log ) 318 (3.80), 287 (3.75) nm; IR
-1 1 (film) vmax 3336, 1704, 1652, 1593, 1514 cm ; H NMR (400 MHz, MeOD) H 2.74 (2H, t, J = 7.4 Hz, H-7), 3.46 (2H, t, H-8), 3.85 (3H, s, OCH3), 6.41 (1H, d, J = 15.7 Hz, H-8 ),
6.71 (2H, d, J = 8.4 Hz, H-3; H-5), 6.79 (1H, d, J = 8.2 Hz, H-5 ), 7.01 (1H, dd, J = 8.3,
1.7 Hz, H-6 ), 7.04 (2H, d, J = 8.4 Hz, H-2; H-6), 7.09 (1H, d, J = 1.6 Hz, H-2 ), 7.44
13 (1H, d, J = 15.7 Hz, H-7 ); C NMR (100 MHz, MeOD) C 169.2 (C=O), 156.9 (C-4),
149.7 (C-3 ), 149.2 (C-4), 142.0 (C-7 ), 131.2 (C-1), 130.7 (C-2, C-6), 128.2 (C-1 ), 123.2
(C-6 ), 118.7 (C-8 ), 116.4 (C-5 ), 116.2 (C-3, C-5), 111.5 (C-2 ), 56.3 (OCH3), 42.5 (C-
+ + 8), 35.8 (C-7); HRESIMS m/z 336.1230 [M+Na] (calcd for C18H19NO4 + Na,
336.1212). The purity of 7 -(3 ,4 -dihydroxyphenyl)-N-[4-methoxyphenyl)ethyl]- propenamide (58) was supported using thin-layer chromatography (TLC) with three
68
solvent systems (10:1 chloroform-methanol Rf = 0.22; 20:1 ethyl acetate-methanol, Rf =
0.65, 1:3 hexane-ethyl acetate, Rf = 0.5).
6.7 Characterization of maslinic acid (62)
25 Maslinic acid (62). White powder; [ ]D +45.1 (c 0.1, MeOH); IR (film) vmax
-1 1 3400, 1684, 1514 cm ; H NMR (400 MHz, CDCl3) H 0.96 (3H, s, H-30 ), 1.00 (3H, s,
H-25 ), 1.02 (3H, s, H-29 ), 1.03 (3H, s, H-26 ), 1.06 (1H, m, H-5 ), 1.09 (3H, s, H-
23 ), 1.19 (1H, m, H-21a), 1.20 (1H, m, H-15a), 1.28 (3H, s, C-27 ), 1.29 (3H, s, H-
24 ), 1.30 (1H, m, H-19a), 1.31 (1H, m, H-1a), 1.35 (1H, m, H-7a), 1.41 (1H, m, H-6a),
1.45 (1H, m, H-21b), 1.53 (1H, m, H-7b), 1.58 (1H, m, H-6b), 1.81 (1H, m, H-9 ), 1.81
(1H, m, H-19b), 1.83 (1H, m, H-22a), 1.98 (1H, m, H-11a), 2.01 (1H, m, H-16a), 2.02
(1H, m, H-11b), 2.05 (1H, m, H-22b), 2.13 (1H, m, H-16b), 2.15 (1H, m, H-15b), 2.26
(1H, m, H-1b), 3.31 (1H, dd, J = 13.6, 3.6 Hz, H-18 ), 3.40 (1H, d, J = 9.3 Hz, H-3 ),
4.10 (1H, ddd, J = 11.0, 9.3, 4.1 Hz, H-2 ), 5.48 (1H, bs, H-12); 13C NMR (100 MHz,
CDCl3) C 180.6 (C-28), 145.3 (C-13), 122.9 (C-12), 84.3 (C-3), 69.0 (C-2), 56.3 (C-5),
48.6 (C-9), 48.2 (C-1), 47.1 (C-17), 46.9 (C-19), 42.6 (C-14), 42.4 (C-18), 40.35 (C-4),
40.30 (C-8), 39.0 (C-10), 34.7 (C-21), 33.7 (C-30), 33.6 (C-7), 33.6 (C-19), 31.4 (C-20),
29.8 (C-24), 28.7 (C-15), 26.6 (C-27), 24.4 (C-11), 24.2 (C-29), 24.1 (C-16), 19.3 (C-6),
18.1 (C-23), 17.9 (C-26), 17.3 (C-25); HRESIMS m/z 495.3433 [M+Na]+ (calcd for
C17H9NO3Na, 495.3450). The purity of maslinic acid (62) was verified using thin-layer
chromatography (TLC) with three solvent systems (1:1 hexane-ethyl acetate, Rf = 0.5;
10:1 chloroform-methanol, Rf = 0.4; 70:5:2.5:2.5 ethyl acetate-acetonitrile-methanol-
water, Rf = 0.7). 69
6.8 Characterization of palmitic acid (57)
Palmitic acid (57). White powder; IR (film) vmax 2954, 2916, 2849, 1700
-1 1 cm ; H NMR (400 MHz, CDCl3) H 1.55 (1H, dd, J = 14.5, 3.6 Hz, H-2 ), 1.80 (3H, s,
5-CH3 ), 1.82 (1H, m, H-4 ), 2.00 (1H, dt, J = 14.5, 2.5 Hz, H-2 ), 2.48 (1H, dt, J =
13 14.0, 2.5 Hz, H-4 ), 4.35 (1H, m, H-3 ), 5.71 (1H, s, H-7); C NMR (100 MHz, CDCl3)
C 182.8 (C-6), 172.3 (C-8), 113.3 (C-7), 87.1 (C-5), 67.2 (C-3), 47.6 (C-2), 46.0 (C-4),
+ 36.3 (C-1), 27.4 (5-CH3); HRESIMS m/z 279.2275 [M+Na] (calcd for C16H32O2Na,
279.2300). The purity of palmitic acid (57) was verified using thin-layer chromatography
(TLC) with three solvent systems [20:1 dichloromethane-acetone, Rf = 0.4; 2:1 hexane-
ethyl ether developed twice, Rf = 0.3 (developed twice); 5:1 hexane-ethyl acetate, Rf =
0.4) with each TLC plate visualized with sulfuric acid spray and heat].
6.9 Characterization of (-)-epicatechin (34)
25 (-)-Epicatechin (34). Colorless needles; mp 175-177 °C; [ ]D -51 (c 1.0,
-1 -1 - CHCl3); CD (MeOH, ) 280.0 (-0.66 mdeg•L•mol •cm ) 240.0 nm (1.3 mdeg•L•mol
1 -1 -1 1 •cm ); IR (film) vmax 3420, 1652, 1647, 1400 cm ; H NMR (300 MHz, MeOD) H 2.73
(1H, dd, J = 16.8, 2.7 Hz, H-4 ), 2.86 (1H, dd, J = 16.8, 4.5 Hz, H-4 ), 4.16 (1H, m, H-
3 ), 4.79 (1H, brs, H-2 ), 5.93 (1H, d, J = 2.3 Hz, H-6), 5.95 (1H, d, J = 2.3 Hz, H-8),
6.75 (1H, d, J = 8.0 Hz, H-5 ), 6.79 (1H, dd, J = 8.3, 1.7 Hz, H-6 ), 6.98 (1H, d, J = 2.1
13 Hz, H-2 ); C NMR (75 MHz, MeOD) C 157.8 (C-7), 157.4 (C-5), 157.2 (C-9), 145.78
(C-4 ), 145.61 (C-3 ), 119.4 (C-6 ), 115.9 (C-5 ), 115.2 (C-2 ), 100.0 (C-10), 96.4 (C-6),
95.9 (C-8), 79.7 (C-2), 67.3 (C-3), 29.1 (C-4); HRESIMS m/z 313.0666 [M+Na]+ (calcd for C51H14O6Na, 313.0688). The purity of (-)-epicatechin (34) was supported using thin- 70
layer chromatography (TLC) with three solvent systems ([1:1:0.1 chloroform-methanol-
acetic acid, Rf = 0.7; 3:1 dichloromethane-acetone (2x), Rf = 0.3; 70:5:2.5:2.5 ethyl
acetate-acetonitrile-methanol-water, Rf = 0.5]).
6.10 Characterization of daucosterol ( -sitosterol-3- -D-glycoside) (63)
25 Daucosterol (63). White powder; [ ]D -41.5 (c 0.1, MeOH); IR (film) vmax 3377,
-1 1 2868, 1457, 1376, 1076, 1019 cm ; H NMR (400 MHz, pyridine-d5) H 0.67 (3H, s, H-
18), 0.87 (3H, brs, H-26), 0.89 (3H, brs, H-27), 0.91 (3H, brs, H-29), 0.95 (3H, s, H-19),
1.01 (3H, d, J = 6.4 Hz, H-21), 4.01 (1H, m, H-5 ), 4.09 (1H, m, H-3), 4.32 (1H, t, J = 9.1
Hz, H-3 ), 4.32 (1H, t, J = 8.6 Hz, H-2 ), 4.44 (1H, dd, J = 11.8, 5.0 Hz, H-6 b), 4.59 (1H,
dd, J = 11.6, 1.56 Hz, H-6 a), 5.08 (1H, d, J = 7.6 Hz, H-1 ), 5.36 (1H, t, J = 4.4 Hz, H-6);
13 C NMR (100 MHz, pyridine-d5) C 141.3 (C-5), 122.4 (C-6), 103.1 (C-1 ), 79.1 (C-3 ),
79.0 (C-3), 78.5 (C-5 ), 75.8 (C-2 ), 75.8 (C-2 ), 71.1 (C-4 ), 63.2 (C-6 ), 57.3 (C-14),
56.7 (C-17), 50.8 (C-9), 46.5 (C-24), 42.9 (C-13), 40.4 (C-12), 39.8 (C-4), 37.9 (C-1),
37.4 (C-10), 36.8 (C-20), 34.6 (C-22), 32.66 (C-7), 32.53 (C-8), 30.7 (C-2), 29.92 (C-25),
29.03 (C-16), 26.8 (C-23), 24.9 (C-15), 23.8 (C-28), 21.7 (C-11), 20.47 (H-27), 19.91 (H-
21), 19.68 (H-26), 19.49 (H-19), 12.64 (H-29), 12.46 (H-18); HRESIMS m/z 599.42832
+ + [M+Na] (calcd for C35H60O2Na , 599.42821). The purity of daucosterol (63) was
supported using thin-layer chromatography (TLC) with three solvent systems [10:1:0.1
chloroform-methanol-AcOH (developed three times), Rf =0.2; 5:1 ethyl acetate-methanol,
Rf = 0.7; 70:5:2.5:2.5 ethyl acetate-acetonitrile-methanol-water (developed twice), Rf =
0.4].
71
6.11 Characterization of (-)-loliolide (65)
23 (-)-Loliolide (65). White powder; [ ]D -105 (c 0.1, MeOH); UV (MeOH) max
-1 1 (log ) 295 (3.77), 260 (3.71) nm; IR (film) vmax 3436, 2947, 1724, 1620 cm ; H NMR
(400 MHz, CDCl3) H 1.55 (1H, dd, J = 14.5, 3.6 Hz, H-2 ), 1.80 (3H, s, 5-CH3 ), 1.82
(1H, m, H-4 ), 2.00 (1H, dt, J = 14.5, 2.5 Hz, H-2 ), 2.48 (1H, dt, J = 14.0, 2.5 Hz, H-
13 4 ), 4.35 (1H, m, H-3 ), 5.71 (1H, s, H-7); C NMR (100 MHz, CDCl3) C 182.8 (C-6),
172.3 (C-8), 113.3 (C-7), 87.1 (C-5), 67.2 (C-3), 47.6 (C-2), 46.0 (C-4), 36.3 (C-1), 27.4
+ (5-CH3); HRESIMS m/z 219.07 [M+Na] (calcd for C11H16O3Na, 219.07). The purity of
(-)-loliolide (65) was verified using thin-layer chromatography (TLC) with three solvent
systems (10:1 chloroform-methanol, Rf = 0.4; 20:1 ethyl acetate-methanol, Rf = 0.7; 1:1
hexane-ethyl acetate, Rf = 0.28).
7. Characterization of synthetic compounds
7.1 Synthesis and characterization of (5R, 6R)-6-[(tert)-butoxycarbonyl)amino]-5-
hydroxy-1-heptene (66).
To a solution of oxalyl chloride (1.44 mL, 17 mmol) in dichloromethane (57 mL)
at -78°C was added DMSO (1.6 mL) dropwise. The mixture was stirred for 30 min,
treated with (R)-2-(Boc-amino)-1-propanol (1 g, 5.70 mmol), and stirred for 2 h.
Triethylamine (11 mL, 68 mmol) was added dropwise, and then was warmed to room
temperature, and then diluted with water (10 mL). The layers were separated, with the
organic layer washed with saturated NH4OH (20 mL) and saturated NaCl (20 mL), dried
(Na2SO4), and evaporated to afford a crude aldehyde, which was taken to the next step
72
without purification. To a solution of this crude aldehyde (1.385 g, 8 mmol) in THF (35
mL), was added (3-butenyl)magnesium bromide (0.5 M, 53 mL) dropwise, at 0°C. After
stirring for 8 h at room temperature, and the reaction was poured into an aqueous
saturated NH4OH solution (80 mL). The organic layer was separated, and the aqueous
layer was washed three times with CHCl3 and the combined organic extracts were
washed sequentially with water and brine. After drying over Na2SO4, the solvent was
removed under vacuum and the residue purified by silica gel column chromatography
(hexane-ethyl acetate 10:1) to yield the amino alcohol 66 (0.8 g, 2 steps 61%) as a yellow
25 oil. This compound exhibited: [ ]D +34 (c 0.1, MeOH); IR (film) vmax 3450, 2900,1670,
-1 1 1160 cm ; H NMR (300 MHz, CDCl3) H 1.14 (3H, d, J = 6.7 Hz, 7-CH3), 1.41 (9H, s,
Boc), 1.45 (2H, m, 3-H2), 2.17 (2H, m, 4-H2), 3.40 (1H, bs, N-H), 3.54 (1H, m, H-6),
4.83 (1H, dd, J = 17.1, 8.7 Hz, H-1a), 4.85 (1H, m, H-5), 5.05 (1H, dd, J = 14.5, 8.7 Hz,
+ H-1b), 5.70 (1H, m, H-2); HRESIMS m/z 252.1570 [M + Na] (calcd for C12H23NO3,
252.1576). The purity of 66 was verified using thin-layer chromatography (TLC) with three solvent systems (5:1 hexane-ethyl acetate, Rf = 0.5; 20:1 dichloromethane-acetone,
Rf = 0.6; 5:1 hexane-acetone, Rf = 0.4).
7.2 Synthesis and characterization of (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-
(tert-Butyldimethylsilyl)-1-heptene (67).
To a solution of 66 (0.8 g, 3.4 mmol) in DMF (53 mL) at room temperature, were
added imidazole (0.1 g, 1.7 mmol) and tert-butyldimethylchlorosilane (TBSCl) (0.2 g,
1.0 mmol). After stirring for 3 h, the reaction was quenched with water (10 mL). The
mixture was extracted with hexane-ethyl acetate (5:1), dried over Na2SO4, and the solvent 73
removed under vacuum. The residue was purified by silica gel column chromatography
hexane-ethyl acetate (15:1), to yield the TBS protected alkene 67 (754 mg, 63%), as a
25 colorless oil. This compound exhibited: [ ]D +38 (c 0.1, MeOH); IR (film) vmax 3450,
-1 1 3300, 1716, 1160 cm ; H NMR (300 MHz, CDCl3) H 0.02 (6H, s, TBS-CH3), 0.85 (9H,
s, TBS-tert-butyl), 1.05 (3H, d, J = 6.6 Hz, 7-CH3), 1.38 (6H, s, Boc), 1.53 (2H, m, 3-H2),
2.01 (2H, m, 4-H2), 3.53 (1H, bs, N-H), 3.67 (1H, m, H-6), 4.62 (1H, bd, J = 8.8 Hz, H-
5), 4.86 (1H, d, J = 10.4 Hz, H-1a), 4.95 (1H, d, J = 17.2 Hz, H-1b), 5.72 (1H, m, H-2);
+ HRESIMS m/z 366.2371 [M + Na] (calcd for C18H37NO3SiNa, 366.2440). The purity of
67 was verified using thin-layer chromatography (TLC) with three solvent systems (5:1
hexane-ethyl acetate, Rf = 0.8; 10:1 hexane-acetone, Rf = 0.4; 3:1 hexane-ethyl ether, Rf =
0.5).
7.3. Synthesis and characterization of (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-
(tert-butyldimethylsilyl)-1,2-diol (68).
To a stirred solution of 67 (0.7 g, 2.2 mmol) and N-methylmorpholine-N-oxide
(50% w/w in water, 265 µL, 4.39 mmol) in acetone (10 mL) and THF (10 mL) was added
polymer-bound OsO4 (10 mg/mol in t-BuOH, 96 µL, 0.082 mmol) at room temperature.
After stirring for 3 h, a saturated aqueous Na2SO3 solution was added to the mixture and
extracted with CHCl3. The combined organic layer was washed with brine and dried over
Na2SO4. The solvent was removed under vacuum, and the residue was purified by silica
gel column chromatography using hexane-ethyl acetate (5:1) to yield diol 68 (600 mg,
25 quantitative yield) as a colorless oil. This compound exhibited: [ ]D +45 (c 0.1, MeOH);
-1 1 IR (film) vmax 3500, 2930, 1690 cm ; H NMR (300 MHz, CDCl3) H 0.06 (6H, s, TBS- 74
CH3), 0.89 (9H, s, TBS-tert-butyl), 1.09 (3H, d, J = 6.3 Hz, 7-CH3), 1.43 (6H, s, Boc),
1.48-1.65 (4H, m, 3-H2, 4-H2), 3.38-3.73 (5H, m, H-1a, H-1b, H-5,H-6, N-H), 4.68 (1H,
+ bd, J = 9.2 Hz, H-2); HRESIMS m/z 400.2466 [M + Na] (calcd for C18H39NO5SiNa,
400.2495). The purity of 68 was verified using thin-layer chromatography (TLC) with three solvent systems (1:1 hexane-ethyl acetate, Rf = 0.3; 70:5:2.5:2.5 hexane-
acetonitrile-water-methanol, Rf = 0.7; 5:1 dichloromethane-acetone, Rf = 0.5).
7.4. Synthesis and characterization of (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-
(tert-butyldimethylsilyl)-1-(tert-butyldimethylsilyl)-2-ol (69).
To a solution of 68 (0.6 g, 1.6 mmol) in DMF (16 mL) at room temperature, was
added imidazole (0.2 g, 3.2 mmol) and tert-butyldimethylchlorosilane (TBSCl) (0.2 g,
1.9 mmol). After stirring for 2 h, the reaction was quenched with water (10 mL). The
mixture was extracted with hexane-ethyl acetate (5:1), dried over Na2SO4, and the solvent
removed under vacuum. The residue was purified by silica gel column chromatography
hexane-ethyl acetate (10:1) to yield the TBS protected alkene 69 (500 mg, 65%) as a
25 colorless oil. This compound exhibited: [ ]D +28 (c 0.1, MeOH); IR (film) vmax 3450,
-1 1 3300, 1716, 1160 cm ; H NMR (300 MHz, CDCl3) H 0.08 (12H, s, TBS-CH3), 0.92
(18H, s, TBS-tert-butyl), 1.11 (3H, d, J = 6.7 Hz, 7-CH3), 1.45 (6H, s, Boc), 1.33-1.38
(2H, m, H-4a, H-4b), 1.52 (1H, m, 3-Ha), 2.50 (1H, m, H-3b), 3.37-3.75 (4H, br m, H-1a,
H-1b, H-5, H-6), 4.67 (1H, bd, J = 8.6 Hz, H-2); HRESIMS m/z 514.3342 [M + Na]+
(calcd for C18H37NO3SiNa, 514.3360). The purity of 69 was verified using thin-layer
chromatography (TLC) with three solvent systems (5:1 hexane-ethyl acetate, Rf = 0.7;
20:1 dichloromethane-acetone, Rf = 0.5; 5:1 hexane-acetone, Rf = 0.4). 75
7.5. Synthesis and characterization of (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-
(tert-butyldimethylsilyl)-1-(tert-butyldimethylsilyl)-2-heptanone (70).
A solution of 69 (0.50 g, 1.0 mmol) in dichloromethane (6 mL) was added
dropwise to a stirring solution of PDC (0.535 g, 1.4 mmol) at room temperature. The
mixture was stirred for 12 h, the solvent removed by evaporation, and the residue diluted
with ethyl acetate. The mixture was filtered through a pad of Celite by washing with ethyl
acetate, and purified by silica gel column chromatography using hexane-ethyl acetate
25 (5:1) to afford 70 (300 mg, 67%) as a colorless oil. This compound exhibited: [ ]D +25
-1 1 (c 0.1, MeOH); IR (film) vmax 3364, 2955, 1716, 1160 cm ; H NMR (300 MHz, CDCl3)
H 0.11 (12H, s, TBS-CH3), 0.92 (18H, s, TBS-tert-butyl), 1.11 (3H, d, J = 5.0 Hz, 7-
CH3), 1.46 (6H, s, Boc), 1.71 (2H, m, H-4a, H-4b), 2.56 (2H, t, J = 5.7 Hz, H-3a, H-3b),
3.64 (1H, dt, J = 7.9, 3.2 Hz, H-5), 3.72 (1H, m, H-6), 4.19 (2H, s, H-1a, H-1b), 4.63
(1H, bd, J = 6.40 Hz, H-5); HRESIMS m/z 512.3188 [M + Na]+ (calcd for
C18H37NO3SiNa, 512.3204). The purity of 70 was verified using thin-layer
chromatography (TLC) with three solvent systems (5:1 hexane-ethyl acetate, Rf = 0.7;
20:1 dichloromethane-acetone, Rf = 0.6; 5:1 hexane-acetone, Rf = 0.5).
7.6. Synthesis and characterization of (2R,3R,6R)-3-[(tert)-butyldimethylsilyl)]-2-
methyl-6-(tert-butyldimethylsilyl)piperidine (72).
A solution of 70 (0.3 g, 0.6 mmol) in dichloromethane (6 mL) was stirred under
argon. After cooling to 0°C, 2,6-lutidine (356 µL, 3.1 mmol) was added, followed by
TMS-triflate (443 µL, 2.5 mmol). The solution was warmed to room temperature and
stirred for 1.5 h. Methanol (15 mL) was added, followed by Et3N (3 mL), and the stirring 76 continued overnight. The solution was concentrated under vacuum to afford a crude imine (71), which was taken to the next step without purification. To a solution of crude imine 71 in MeOH (7 mL) was added 10% Pd-C (2 mg) and the mixture was stirred under a H2 atmosphere for 12 h. The reaction mixture was filtered through a pad of
Celite, washing with MeOH. The combined washings were concentrated under vacuum and purified by silica gel column chromatography (hexane-ethyl acetate 10:1), to afford
25 72 (75 mg, two steps 33%), as a yellow oil. This compound exhibited: [ ]D -41.7 (c 0.1,
-1 1 MeOH); IR (film) vmax 3200, 3150, 2955 cm ; H NMR (300 MHz, CDCl3) H 0.04
(12H, s, TBS-CH3), 0.87 (9H, s, TBS-tert-butyl), 0.89 (9H, s, TBS-tert-butyl), 0.99 (3H, d, J = 6.6 Hz, CH3-2 ), 1.28 (1H, m, H-5 ), 1.56 (1H, m, H-4 ), 1.73 (1H, m, H-5 ),
1.86 (1H, m, H-4 ), 2.57 (1H, m, H-6 ), 2.68 (1H, dq, J = 12.6, 6.1 Hz, H-2 ), 3.53 (1H,
13 m, H-3 ), 3.56-3.62 (2H, m, H-7a, H-7b); C NMR (75 MHz, CDCl3) C 19.4 (CH3-2),
21.9 (C-5), 32.5 (C-4), 55.2 (C-2), 57.4 (C-6), 67.1 (C-7), 68.1 (C-3); HRESIMS m/z
+ 396.2716 [M + Na] (calcd for C18H37NO3SiNa, 396.2730). The purity of 72 was verified using thin-layer chromatography (TLC) with three solvent systems (4:1:0.05 ethyl ether- acetonitrile-ammonium hydroxide, Rf = 0.6; 15:1:0.05 chloroform-methanol-ammonium hydroxide, Rf = 0.5; 3:1:0.05 ethyl acetate-acetonitrile-ammonium hydroxide, Rf = 0.7).
8. Cytotoxicity and nAChR antagonistic activity of isolated alkaloids from M. paniculata and synthetic analogues
All isolated compounds from active fractions obtained from the stem bark, branches and leaves of M. paniculata were evaluated for their cytotoxicity against HT-29 human colon cancer cells. The effects of 56, 58-61, 64, 72 and the C-3 acetate of 77 compound 56 were also tested using a functional calcium accumulation assay with
HEKtsA201 cells stably expressing either human h 4 2 or h 3 4 neuronal nicotinic acetylacholine receptors (nAChRs). When assayed at a single concentration (10 µM), the inhibition of epibatidine-stimulated calcium accumulation on h 4 2 or h 3 4 nAChRs was quantified (Table 3.5). Calcium accumulation assays were also performed to obtain dose-response IC50 values (Table 3.6). The competitive or non-competitive nature of the inhibitory activity was investigated by determining the effects of increasing concentrations of epibatidine in the absence and presence of microgrewiapine A (56).
The effect of epibatidine in the presence of 56 was not surmountable with increasing concentrations of epibatidine (maximum effect of 41.2 ± 3.3 %), compared to this agonist alone (95.1 ± 5.6 %) (Table 3.5).
78
HT-29a CCD-112CoNb methanol extract (stem bark) >20 NTc methanol extract (branches) >20 NTc methanol extract (leaves) 13.2 NTc hexane-soluble extract (stem bark) >20 NTc hexane-soluble extract (branches) >20 NTc hexane-soluble extract (leaves) >20 NTc chloroform-soluble extract (D3) (stem bark, A6319*) 9.9 NTc chloroform-soluble extract (D3) (branches, A6320*) 13.1 NTc chloroform-soluble extract (D3) (leaves, A6317*) 2.9 NTc A6319D3F6* 6.0 NTc A6320D3F7* 11.6 NTc A6320D3F8* 14.3 NTc A6317D3F6* 10.1 NTc A6317D3F8* 12.9 NTc A6317D3F9* 11.5 NTc A6317D3F10* 13.4 NTc A6317D3F11* 1.7 NTc microgrewiapine A (56) 6.8d 30.4d microgrewiapine 3-acetate 14.0d NTc 7 -(3 ,4 -dihydroxyphenyl)-N- [4- methoxyphenyl)-ethyl]propenamide (58) 72.7d NTc liriodenine (59) 25.7 d NTc microgrewiapine B (60) >20 NTc microgrewiapine C (61) >20 NTc (2R,3R,6R)-3-[(tert)-butyldimethylsilyl)]-2-methyl-6- (tert-butyldimethylsilyl)piperidine (72) 13.6 d NTc 1,3-dimethylpiperidine (73) >20 NTc piperidin-3-ol (74) >20 NTc paclitaxele 0.006d 23.0d aCytotoxicity of the major subfractions from each plant part are shown. bNon-cancerous colon cell line. cNT = not tested. d Results for pure compounds are expressed as IC50 values (µM), all other values are expressed as µg/mL. eUsed a positive control substance. *Refer to the program project code-number (A number), D3 to chloroform soluble extract, “F” and “R” signify subfraction numbers, “K” signifies a pure compound. Table 3.4 Cytotoxicity assay results of Microcos paniculata extracts, fractions, and pure compounds against HT-29 human colon cancer cells. Pure compounds exhibiting IC50 values of <10 µM were also evaluated against the CCD-112CoN non-cancerous colon cell line to ascertain cancer cell selectivity.
79
h 4 2 nAChR h 3 4 nAChR compound % inhibitiona,b % inhibitiona,b microgrewiapine A (56) 74.0 ± 14.2 58.2 ± 9.2 microgrewiapine B (60) 79.3 ± 6.0 82.7 ± 3.3 microgrewiapine C (61) 67.3 ± 28.0 82.8 ± 11.5 microcosamine A (64) 59.0 ± 7.8 53.0 ± 9.5 7 -(3 ,4 -dihydroxyphenyl)-N- [4- methoxyphenyl)-ethyl] propenamide (58) 21.8 ± 6.2 14.0 ± 6.6 liriodenine (59) 13.3 ± 24.5 19.3 ± 24.4 microgrewiapine A 3-acetate 44.1 ± 22.3 NTc D-tubocurarined 61.0 ± 27.9 54.0 ± 23.5 mecamylamined 91.8 ± 5.6 88.3 ± 13.3 KAB-18d 39.0 ± 11.7 NAe epibatidinef 41.2 ± 3.3f NTf aPercent inhibition was determined at 10 µM. bValues represent means ± SD, n = 2-5. cNT = not tested. dKnown nicotinic receptor antagonist. eNA = no activity at 10 µM. fMaximum effect of 1 µM epibatidine in the presence of 10 µM 56. Table 3.5 Percent inhibition of nAChR activity by test compounds applied at a 10 µM dose.
h 4 2 nAChR nH h 3 4 nAChR nH a a compound IC50(µM) (CL) IC50(µM) (CL) microgrewiapine A (56) 4.6 (3.2-6.7) -1.3 8.3 (7.1-9.7) -1.2 microgrewiapine B (60) 2.6 (2.0-3.4) -0.8 2.9 (1.9-4.6) -1.2 microgrewiapine C (61) 4.0 (1.4-11.1) -1.0 2.4 (1.1-5.4) -1.0 liriodenine (59) 89.8 (70.8-114.0) -0.9 >100 ~ 7 -(3 ,4 -dihydroxyphenyl)-N- -1.2 -1.2 [4-methoxyphenyl)- ethyl]propenamide (58) 81.0 (64.3-102.0) 51.9 (37.6-71.4) microcosamine A (64) 10.9 (4.9-24.5) -0.9 NTb NTb microgrewiapine A 3-acetate 10.6 (8.0-14.1) -0.8 NTb NTb D-tubocurarinec 2.3 (1.9-3.9) -0.7 1.6 (0.4-3.5) -0.9 (2R,3R,6R)-3-[(tert)- -0.9 -0.9 butyldimethylsilyl)]- 2-methyl-6-(tert- butyldimethylsilyl) piperidine (72) 21.0 (14.0-31.7) 22.4 (13.1-38.1) 1,3-dimethylpiperidine (73) N/Ad N/Ad piperidin-3-ol (74) N/Ad N/Ad mecamylaminec 0.5 (0.3-0.8) -0.9 0.2 (0.1-0.3) -0.8 KAB-18c 13.5 (9.7-18.5) -0.7 N/Ad ~ (-)-nicotine (18)e 8.9 (5.7-13.9) 1.2 9.1 (4.5-18.6) 0.9 epibatidinee 18.1 (6.5-50.6) nM 0.6 37.4 (10.7-71.4) nM 0.9 aValues represent geometric means (confidence limits), n = 2-8. bNT = not tested. cKnown nicotinic receptor d e antagonist. NA = no activity at concentrations up to 100µM. nH, Hill coefficient. Value represents EC50. Table 3.6 Effects of isolated and synthesized antagonists on human nAChRs.
80
C. Discussion
1. General points on the characterization of the compounds isolated from M.
paniculata
The 2,3,6-trisubstituted piperidine alkaloids isolated in this investigation were
found to be very similar in chemical structure, in some cases having the same molecular
weight (m/z) but differing only in the configuration at a single carbon atom. A number of
diagnostic 1H and 13C NMR chemical shifts were apparent in the NMR spectra of this
class of compounds. The piperidine ring protons attached to C-2, C-3 and C-6 typically
appeared as a 3H doublet, a 1H doublet of triplets (6H), and a 1H doublet of doublets of
doublets (3H), at approximately 1.5, 2.5, and 3.5 ppm, respectively, in the 1H NMR spectrum. The corresponding 13C NMR signals for C-2, C-3 and C-6 appeared at
approximately 65, 72, and 67 ppm, respectively. When the piperidine ring nitrogen atom
1 was N-methylated, the signal (N-CH3) appeared at approximately 40 ppm. The H and
13C NMR signals were shifted downfield in the presence of an N-oxide moiety at the
piperidine ring nitrogen atom by approximately 5 ppm. The numbering system used for
the isolated piperidine alkaloids is given in Figure 3.5, using microgrewiapine A (56) as
an example.
Figure 3.5 Numbering system used for piperidine alkaloids isolated from Microcos paniculata. 81
2. Structure elucidation of microgrewiapine A (56)
Microgrewiapine A (56) was obtained as colorless needle crystals, mp 127-128
15 °C, [ ]D +15.4 (c 0.1, MeOH). A molecular formula of C17H29NO was determined from
+ + the molecular ion peak at m/z 264.2329 [M + H] (calcd for C17H29NO + H , 264.2327) in
the HRESIMS, which indicated the presence of a single nitrogen atom in the molecule.
The IR spectrum showed bands at 3402, 2929, and 1629 cm-1, suggestive of hydroxy
group absorption and olefinic C-H stretching, respectively. The UV spectrum exhibited a
maximum at 270 nm, consistent with conjugation in the molecule. The 1H NMR and 13C
NMR spectra (600 MHz, CDCl3) showed signals for six methine groups in the region H
5.53-6.16, along with resonances at H 2.09 (2H, dt, J = 7.2, 6.9 Hz, H-7 ), 1.29-1.38 (4H,
m, H-8 , H-9 ) and 0.89 (3H, t, J = 7.2 Hz, H-10 ), which correlated in the HSQC
spectrum to six olefinic methine carbons ( C 130.1-132.6), three methylene carbons [ C
32.5 (C-7 ); 31.4 (C-8 ); 22.0 (C-9 )], and a methyl carbon [ C 14.1 (C-10 )], respectively.
These NMR signals indicated the presence of a deca-1E,3E,5E-trienyl group in the
molecule of 56. The remaining 1H and 13C NMR signals were attributed to a methyl
group [ H 1.26 (3H, d, J = 6.1 Hz, CH3-2); C 16.4], two methylene groups [ H 2.03 (1H, m, H-4 ), 1.29-1.38 (1H, m, H-4 ), 1.63 (1H, m, H-5 ), 1.49 (1H, ddd, J = 10.1, 3.0, 3.0
Hz, H-5 ); C 33.5 (C-4), 31.3 (C-5)], an oxygen-bearing methine group [ H 3.27 (1H,
ddd, J = 10.8, 8.9, 4.5 Hz, H-3 ); C 72.6 (C-3)], two nitrogen-bearing methine groups
[ H 2.48 (1H, ddd, J = 11.2, 3.0 Hz, H-6 ), 1.83 (1H, dq, J = 8.9, 6.1 Hz, H-2 ); C 67.6
(C-6), 66.1 (C-2)], and a N-CH3 group at H 2.21 (3H, s, N-CH3); C 40.4. The oxygen-
bearing methine group at H 3.27 (H-3) showed a COSY correlation with the nitrogen- 82
bearing methine group at H 1.83 (H-2). The methylene signals H 1.49 and H 1.63, corresponding to the C-5 protons, exhibited a COSY correlation with the nitrogen-
1 13 bearing methine group at H 2.48 (C-6) (Figure 3.16). A heteronuclear H C HMBC three-bond correlation between 2-CH3 ( H 1.26) and C-3 ( C 72.6), a four-bond
correlation between the N-CH3 ( H 2.21) group and 2-CH3, and a two-bond correlation
between both C-5 protons and the nitrogen-bearing methine group at H 2.48 (C-6), were
observed. Taken together, the chemical shift values and connectivity patterns from the
COSY and HMBC spectra indicated the presence of a N-methyl-2-methyl-3-piperidinol
1 ring (Bandara et al., 2000; Feng et al., 2008a; Luo et al., 2009). The H NMR signal at H
5.53 (1H, dt, J = 14.6, 8.8 Hz, H-1 ) was coupled to the nitrogen-bearing methine signal
at H 2.48 (C-6) with a magnitude of 8.7 Hz, and also showed a COSY correlation to the
same methine proton, indicating that the deca-1E,3E,5E-trienyl group is attached to the
N-methyl-2-methyl-3-piperidinol ring at C-6. The double bond geometry at 1 ,2 and 5 ,6 was determined to be trans in each case, based on the large coupling constants of J1 2 =
3 ,4 14.6 Hz and J5 6 = 14.5 Hz observed. For the double bond, the configuration could
not be determined by coupling constants due to signal overlap. Differences in 1H and 13C
chemical shifts between the (1 E,3 E,5 E) and (1 E,3 Z,5 E) geometries, however, could
be used to elucidate the 3 ,4 configuration. If the conjugated triene system adopts a
(1 E,3 E,5 E) geometry, the NMR chemical shifts of H-2 /H-5 and C-2 /C-5 are typically
around H 6.0 ppm and C 130 ppm, respectively (Ando et al., 1988). If the conjugated
triene instead adopts a (1 E,3 Z,5 E) geometry, H H-2 /H-5 shifts ~0.4 ppm downfield,
and C C-2 /C-5 shifts ~5 ppm upfield (Ando et al., 1988). The triene system of 56
83 showed chemical shifts of H 6.03-6.16 (H-2 , H-5 ) and C 130.1-132.6 (C-2 , C-5 ), which supported a trans configuration for 3 ,4 . The absolute configuration at the C-3 hydroxy group position was accomplished using a Mosher esterification procedure
(Reiser et al., 1992). Two portions of 56 (each 1.0 mg) were treated with (S)-(+)- - and
(R)-(-)- -methoxy- -(trifluoromethyl)phenylacetyl chloride (14 µL) in deuterated pyridine (0.5 mL) directly in separate NMR tubes at room temperature, affording the (R)- and (S)-MTPA esters, respectively (Reiser et al., 1992; Su et al, 2002; Seco et al., 2004).
Observed chemical shift differences ( S-R) indicated the absolute configuration at the
OH-3 group of 56 to be R.
With the absolute configuration at the OH-3 position determined, analysis of coupling constants and NOESY correlations was used to establish the relative configuration of the remaining substituents of the piperidinol ring. The 3R absolute configuration indicated that H-3 ( H 3.27) is oriented on the -face of the piperidine ring.
The coupling constant between H-3 , and the signal at H 1.83 (H-2 ) was 8.9 Hz, indicating a trans relationship. Strong NOESY correlations between H-2 and the signal at H 2.48 (H-6) suggested that the latter proton is also oriented on the -face of the piperidinol ring. Accordingly, microgrewiapine A (56) was assigned structurally as
(2S,3R,6S)-6-[(1E,3E,5E)-deca-1,3,5-trien-1-yl]-1,2-dimethylpiperidin-3-ol.
The 1D- and 2D-NMR spectra obtained for this compound are presented in
Figures 3.6-3.17.
84
1.64 - 1.50 = +0.14 1.91 - 1.89 = +0.02 OH 2.42 - 2.37 = +0.05 H 1.12 - 1.25 = -0.13 CH3 N H
CH3 2.56 - 2.60 = -0.04
Figure 3.6. Chemical shift difference values (S-R), for protons on the esterified piperidinol ring of 56, as established by a modified Mosher ester method.
1 Figure 3.7. H NMR spectrum of microgrewiapine A (56) (CDCl3, 600 MHz)
85
1 Figure 3.8. H NMR spectrum of microgrewiapine A (56) (CDCl3, 600 MHz; expanded scale)
13 Figure 3.9. C NMR spectrum of microgrewiapine A (56) (CDCl3, 150 MHz) 86
13 Figure 3.10. C NMR spectrum of microgrewiapine A (56) (CDCl3, 150 MHz; expanded scale)
Figure 3.11. DEPT spectrum of microgrewiapine A (56) (CDCl3)
87
Figure 3.12. DEPT spectrum of microgrewiapine A (56) (CDCl3; expanded scale)
Figure 3.13. HSQC spectrum of microgrewiapine A (56) (CDCl3)
88
Figure 3.14. HSQC spectrum of microgrewiapine A (56) (CDCl3; expanded scale)
Figure 3.15. HMBC spectrum of microgrewiapine A (56) (CDCl3)
89
Figure 3.16. COSY spectrum of microgrewiapine A (56) (CDCl3, 600 MHz)
Figure 3.17. NOESY spectrum of microgrewiapine A (56) (CDCl3, 600 MHz). 90
3. Structure elucidation of microgrewiapine B (60)
Microgrewiapine B (60) was obtained as a clear amorphous solid, mp 134-136
25 °C, [ ]D 4.0 (c 0.1, MeOH). A molecular formula of C17H29NO2 was determined from
+ + the molecular ion peak at m/z 280.2269 [M + H] (calcd for C17H29NO2 + H , 280.2276)
in the HRESIMS. The IR spectrum showed absorption bands at 3400, 2967, 1670, 1204,
and 1000 cm-1 indicative of the presence of hydroxy group, olefinic C-H, C-N, and
aliphatic amine oxide functionalities, respectively. The 1H NMR and 13C NMR spectra
(400 MHz, CDCl3) showed that signals in the piperidine ring were shifted downfield,
when compared to 56, due to the presence of a N-oxide moiety. Downfield shifts were
pronounced in both the nitrogen-bearing methine signals at H 2.86 (1H, dq, J = 10.7,
6.12 Hz, H-2 ) and 3.44 (1H, m, H-6 ), and the oxygen-bearing methine signal at H 3.90
(1H, ddd, J = 11.2, 10.5, 4.8 Hz, H-3 ). Despite the downfield shifts of these signals, the splitting patterns were comparable to the analogous signals in compound 56 (Figure
3.19). The coupling constant between the oxygenated methine at H 3.90 (H-3), and the
signal at H 2.86 (H-2) was 10.7 Hz, indicating a trans relationship, with H-3 and H-2
oriented axially on the piperidinol ring.
Two configurations at the nitrogen atom of 60 are possible. For a 1R
configuration, a NOESY correlation between N-CH3 and H-3 would not be expected.
Indeed, no NOE correlation was observed between the signals at H 2.95 (N-CH3) and H
3.90 (H-3) (Figure 3.18). Strong NOESY correlations were seen between the N-methyl
( H 2.95) and H-2 ( H 2.86) signals, due to the proximity within 4 Å of these
91 functionalities. In addition, strong NOESY correlations were observed between the signals at H 2.86 (H-2) and 3.44 (1H, m, H-6), indicating these protons to be located on the same face of the piperidinol ring. The 1H NMR coupling patterns and NOESY correlations observed for 60 were supported by in silico MP2 energy minimization studies using the 6-311 G (d,p) basis set, implemented in the Guassian 09 program
(Gaussian 09, Rev. A.01). Relative energies of the 1R and 1S enantiomers of 59 in the gas phase, ethanol, and water were computed and the 1R enantiomer was found to be the more energetically favorable. Energy difference values between the 1R and 1S enantiomers of 59 were calculated to be 2.76, 1.08, and 0.94 kcal/mol, for the gas phase, ethanol, and water, respectively. The solvent effects were calculated with a polarized continuum model that implicitly calculates the effects the solvent has on the molecule.
Microgrewiapine B (60) was assigned structurally, therefore, as (2S,3R,6S)-6-
[(1E,3E,5E)-deca-1,3,5-trien-1-yl]-3-hydroxy-1,2-dimethylpiperidine 1-oxide.
The 1D- and 2D-NMR spectra obtained for this compound are presented in
Figures 3.18-3.26.
Figure 3.18. Selected NOESY correlations of microgrewiapine B (60) (left) and energy- minimized structure (right).
92
1 Figure 3.19. H NMR spectrum of microgrewiapine B (60) (CDCl3, 400 MHz).
13 Figure 3.20. C NMR spectrum of microgrewiapine B (60) (CDCl3, 100 MHz).
93
Figure 3.21. DEPT spectrum of microgrewiapine B (60) (CDCl3).
Figure 3.22. HSQC spectrum of microgrewiapine B (60) (CDCl3).
94
Figure 3.23. HSQC spectrum of microgrewiapine B (60) (CDCl3, expanded scale)
Figure 3.24. HMBC spectrum of microgrewiapine B (60) (CDCl3) 95
Figure 3.25. COSY spectrum of microgrewiapine B (60) (CDCl3)
Figure 3.26. NOESY spectrum of microgrewiapine B (60) (CDCl3)
96
4. Structure elucidation of microgrewiapine C (61)
Microgrewiapine C (61) was obtained as colorless needle crystals, mp 130-131
25 °C, [ ]D +77.8 (c 0.1, MeOH). A molecular formula of C17H29NO2 was determined
+ + from the molecular ion peak at m/z 280.2281 [M + H] (calcd for C17H29NO2 + H ,
280.2277) in the HRESIMS. The UV and IR spectra were closely comparable to those of
1 the 3R enantiomer, compound 60. The H NMR signal at H 3.82 (1H, bs, H-3) appeared
as a broad singlet when compared to the ddd pattern as observed for 60. The methine
signal at H 3.05 (1H, dq, J = 12.7, 6.5 Hz, H-2) would be expected to cause a split
pattern for H-3, but no peak multiplicity was observed for this proton. The 3S
configuration was supported by strong NOESY correlations between the signals at H
3.05 (1H, dq, J = 12.7, 6.5 Hz, H-2), 3.82 (1H, bs, H-3), and 3.53 (1H, ddd, J = 11.4, 9.4,
1.9 Hz, H-6), indicating that these substituents are all on the same face of the piperidinol
ring. In addition, a strong NOESY correlation between H 2.86 (3H, s, N-CH3) and H
3.82 (1H, bs, H-3) was observed, confirming the opposite configuration from 60, for which this correlation was absent (Figure 3.34). Microgrewiapine C (61) was therefore assigned structurally as (2S,3S,6S)-6-[(1E,3E,5E)-deca-1,3,5-trien-1-yl]-3-hydroxy-1,2- dimethylpiperidine 1-oxide.
The 1D- and 2D-NMR spectra obtained for this compound are presented in
Figures 3.27-3.34.
97
1 Figure 3.27. H NMR spectrum of microgrewiapine C (61) (CDCl3, 400 MHz)
13 Figure 3.28. C NMR spectrum of microgrewiapine C (61) (CDCl3, 100 MHz)
98
Figure 3.29. DEPT spectrum of microgrewiapine C (61) (CDCl3)
Figure 3.30. HSQC spectrum of microgrewiapine C (61) (CDCl3) 99
Figure 3.31. HSQC spectrum of microgrewiapine C (61) (CDCl3; expanded scale)
Figure 3.32. HMBC spectrum of microgrewiapine C (61) (CDCl3)
100
Figure 3.33. COSY spectrum of microgrewiapine C (61) (CDCl3)
Figure 3.34. NOESY spectrum of microgrewiapine C (61) (CDCl3)
101
5. Identification of microcosamine A (64)
25 Microcosamine A (64) was obtained as a clear amorphous solid, [ ]D 4.0 (c 0.1,
MeOH). A molecular formula of C16H27NO was determined from the molecular ion peak at
+ 1 m/z 250.2163 [M + H] (calcd for C17H30NO, 250.2171) in the HRESIMS. The H NMR and
13 C NMR spectra (600 and 150 MHz, respectively, CDCl3) showed signals for six methine groups in the region H 5.64-6.20, along with resonances at H 2.10 (2H, dt, J = 6.9, 6.4 Hz,
H-7 ), 1.27-1.39 (4H, H-8 , H-9 ) and 0.90 (3H, t, J = 7.2 Hz, H-10 ), which correlated in the
HSQC spectrum to olefinic methine carbons in the region [ C 131.0-137.7], three methylene
carbons [ C 33.5 (C-7 ); 32.4 (C-8 ); 23.2 (C-9 )], and a methyl carbon [ C 14.9 (C-10 )],
respectively. These NMR signals indicated the presence of a deca-trienyl group in the
molecule of 64. Double bond geometry at 1 ,2 and 5 ,6 was determined to be trans based on the large coupling constants J1 2 = 15.1 Hz and J5 6 = 15.1 Hz observed. As with microgrewiapines A-C (55, 59, 60), the 3 ,4 configuration could not be determined by coupling constants due to signal overlap. Differences in 1H- and 13C-NMR chemical shifts between the possible (1 E,3 E,5 E) and (1 E,3 Z,5 E) geometries were used, however, to
elucidate 3 ,4 as being trans configured. When the conjugated triene system adopts the
(1 E,3 E,5 E) geometry, the NMR chemical shifts of H-2 /H-5 and C-2 /C-5 are typically
around H 6.0 ppm and C 130 ppm, respectively (Ando et al., 1988). If the conjugated triene
instead adopts a (1 E,3 Z,5 E) geometry, H H-2 /H-5 shifts ~0.4 ppm downfield, and C C-
2 /C-5 shifts ~5 ppm upfield (Ando et al., 1988). The triene system of 64 showed chemical
shifts of H 6.02-6.28 (H-2 , H-5 ) and C 131.0-137.7 (C-2 , C-5 ), which supported a trans
configuration for 3 ,4 . The remaining 1H- and 13C-NMR signals were attributed to a methyl
group [ H 1.32 (3H, m, 2-CH3); C 19.3], two methylene groups [ H 1.27-1.32 (1H, m, H-4 ), 102
1.45 (1H, m, H-4 ), 1.55-1.65 (1H, m, H-5 ), 1.80 (1H, m, H-5 ); C 33.5 (C-4), 32.4 (C-5)], an oxygen-bearing methine group [ H 3.36 (1H, m, H-3); C 74.4 (C-3)], and two nitrogen-
bearing methine groups [ H 3.35 (1H, m, H-6), 2.69 (1H, m, H-2); C 59.8 (C-6), 59.4 (C-2)].
1 The N-CH3 signal observed in the H NMR spectra of microgrewiapines A-C [typically around H 2.21 (3H, s, N-CH3); C 40.4] was absent, supporting a free amine moiety in 64.
The oxygen-bearing methine group resonance at H 3.36 (H-3) showed a COSY correlation
with the nitrogen-bearing methine group signal at H 2.69 (H-2). The methylene signals H
1.55-1.65 and H 1.80, corresponding to the C-5 protons, exhibited a COSY correlation with
1 13 the nitrogen-bearing methine group at H 3.35 (C-6). A heteronuclear H C HMBC three- bond correlation between CH3-2 ( H 1.32) and C-3 ( C 74.4), a two-bond correlation between
CH3-2 ( H 1.32) and C-2 ( C 59.4), and a three-bond correlation between H 1.27-1.32 (1H, m,
H-4 ) and the nitrogen-bearing methine group at H 3.35 (C-6), were observed. These chemical shift values and connectivity patterns from the COSY and HMBC spectra indicated the presence of a N-methyl-2-methyl-3-piperidinol ring (Bandara et al., 2000; Feng et al.,
1 2008a; Luo et al., 2009). The H NMR signal at H 5.65 (1H, dd, J = 15.1, 7.4 Hz, H-1 )
showed a COSY correlation to the nitrogen-bearing methine signal at H 3.36 (C-6) indicating
that the deca-1E,3E,5E-trienyl group is attached to the N-methyl-2-methyl-3-piperidinol ring
at C-6. The relative configuration of 64 was not ascertained through NOESY correlation
analysis due to low sample amount (<0.6 mg) obtained. However, the optical rotation value
25 [ ]D 4.0 (c 0.1, MeOH) closely matched that reported in the literature for the same
compound (Feng et al., 2008a). Accordingly, compound 64 was identified as the known
compound, microgrewiapine A {(2S,3R,6S)-6-[(1E,3E,5E)-deca-1,3,5-trien-1-yl]-2-
methylpiperidin-3-ol}.
103
The 1D- and 2D-NMR spectra obtained for this compound are presented in
Figures 3.35-3.39.
1 Figure 3.35. H NMR spectrum of microcosamine A (64) (CDCl3, 600 MHz)
Figure 3.36. DEPT spectrum of microcosamine A (64) (CDCl3) 104
Figure 3.37. HSQC spectrum of microcosamine A (64) (CDCl3)
Figure 3.38. HMBC spectrum of microcosamine A (64) (CDCl3)
105
Figure 3.39. COSY spectrum of microcosamine A (64) (CDCl3)
6. Identification of liriodenine (59)
Liriodenine (59) was obtained as a yellow amorphous powder, mp 276-278 °C. A molecular formula of C17H9NO3 was determined from the molecular ion peak at m/z
+ 298.0507 [M + Na] (calcd for C17H9NO3Na, 298.0480) in the HRESIMS, which indicated the presence of a single nitrogen atom in the molecule. The IR spectrum showed bands at 2930, 1650, and 1580 cm-1, suggestive of olefinic C-H stretching, carbonyl stretching, and aromatic C=C stretching, respectively. The UV spectrum exhibited maxima at 408, 296, and 270 nm consistent with conjugation in the molecule.
The 1H NMR spectrum analyzed in conjunction with the COSY and HSQC spectra showed coupled signals at H 7.75 (1H, d, J = 4.9 Hz, H-4) and 8.87 (1H, d, J =
106
5.0 Hz, H-5) with these assigned to C-4 (124.7) and C-5 (145.3), respectively. Of these
coupled B ring protons, the signal at H 8.87 (H-5) was deshielded as a result of the
proximity to the C-6a imine moiety. Four D-ring aromatic protons at H 8.56 (1H, d, J =
7.8 Hz, H-8), 7.56 (1H, t, J = 7.5, H-9), 7.71 (1H, t, J = 4 Hz, H-10), and 8.60 (1H, d, J =
8.1 Hz, H-11), correlated with carbon signals at C 129.2 (C-8), 128.9 (C-9), 134.3 (C-
10), and 127.7 (C-11) in the HSQC spectrum. Of the ring D protons, H-8 ( H 8.56), in particular, exhibited a three-bond HMBC correlation to the C-7 carbonyl moiety ( C
182.8), and was deshielded by anisotropic effects of the attached quinolone unit
(Sobarzo-Sánchez et al., 2003). The methylenedioxy signal at [ H 6.36 (2H, s); C 102.9] showed a three-bond HMBC correlation to C 152.0 (C-1) and 148.3 (C-2) supporting the
connectivity of this functionality to ring B of the oxoaporphine skeleton. This was
corroborated by a HMBC two-bond correlation between the signal at H 7.17 (1H, s, H-3)
to C-1 and C-2. The physical and spectroscopic data were consistent with literature
values for the known compound, liriodenine (59) (Harrigan et al., 1994; Sobarzo-Sánchez
et al., 2003).
The 1D- and 2D-NMR spectra obtained for this compound are presented in
Figures 3.40-3.45.
107
Figure 3.40. Proton spectrum of liriodenine (59) (CDCl3, 400 MHz)
13 Figure 3.41. C NMR spectrum of liriodenine (59) (CDCl3, 100 MHz)
108
Figure 3.42. DEPT spectrum of liriodenine (59) (CDCl3)
Figure 3.43. HSQC NMR spectrum of liriodenine (59) (CDCl3)
109
Figure 3.44. HMBC NMR spectrum of liriodenine (59) (CDCl3)
Figure 3.45. COSY NMR spectrum of liriodenine (59) (CDCl3)
110
7. Identification of 7 -(3 ,4 -dihydroxyphenyl)-N-[4-methoxyphenyl)ethyl]
propenamide (58).
7 -(3 ,4 -Dihydroxyphenyl)-N-[4-methoxyphenyl)ethyl]propenamide (58) was
obtained as a colorless solid, mp 101-102°C. A molecular formula of C18H19NO4 was
determined from the molecular ion peak at m/z 336.1230 [M+Na]+ (calcd for
C18H19NO4Na, 336.1212) in the HRESIMS, which indicated the presence of a single
nitrogen atom in the molecule. The IR spectrum showed bands at 3336, 1704, 1652,
1593, and 1514 cm-1, supporting a olefinic C-H, carbonyl, aromatic C=C, and C=N
stretching, respectively. The UV spectrum exhibited maxima at 318 and 287 nm,
1 suggesting conjugation in the molecule. The H NMR spectrum showed a signal at H
3.85 (3H, s, 4-OCH3), with a three-bond HMBC correlations to the signal at C 149.23
(C-4). A pair of doublets at H 6.71 (2H, d, J = 8.4 Hz, H-3; H-5) and H 7.04 (2H, d, J =
8.4 Hz, H-2; H-6) exhibited COSY correlation between each other along with a HMBC
correlation to C 149.2 (C-4). A quaternary carbon signal at C 131.2 (C-1) exhibited a
three-bond HMBC correlation to the C-2/C-6 methine protons, in addition to a pair of
methylene protons at H 2.74 (2H, t, J = 7.4 Hz, H-7) and H 3.46 (2H, t, J = 7.2 Hz, H-8).
The diastereotopic H-7/H-8 protons were correlated to each other in the COSY spectrum.
In conjunction with the ESIMS suggesting one nitrogen atom in the moleule of 58, the
aforementioned NMR correlations and signals were consistent with the presence of a
methylated tyramine unit. The remaining methine signals at H 7.44 (1H, d, J = 15.7 Hz,
H-7 ; C 142.0), 6.41 (2H, d, J = 15.7 Hz, H-8 ; C 118.7), 7.09 (1H, d, J = 1.6 Hz, H-2 ;
C 111.5), 7.01 (1H, dd, J = 8.3, 1.7 Hz, H-6 ; C 123.2), and 6.79 (1H, d, J = 8.2 Hz, H-
111
5 , C 116.4) were attributed to a feruloyl unit in the molecule of 58. The methine signals
for H-2 , H-5 and H-6 exhibited HMBC correlations to the quaternary carbon signals at
C 128.2 (C-1 ), 149.7 (C-3 ) and 149.2 (C-3 ). The connection between feruloyl and tyramine moieties in the molecule of 58 was made apparent from the signal at H 3.46 (H-
8) exhibiting a HMBC correlation to the signal at C 169.1 (C=O). The physical and spectroscopic data were consistent with literature values for the known compound, 7 -
(3 ,4 -dihydroxyphenyl)-N-[4-methoxyphenyl)ethyl]propenamide (58) (Woo et al., 1997;
Anis et al., 2002).
The 1D- and 2D-NMR spectra obtained for this compound are presented in
Figures 3.46-3.52.
Figure 3.46. 1H NMR (400 MHz) spectrum of 7 -(3 ,4 -dihydroxyphenyl)-N-[4- methoxyphenyl)ethyl]propenamide (58) (MeOD, 400 MHz)
112
Figure 3.47. 13C NMR spectrum (400 MHz) of 7 -(3 ,4 -dihydroxyphenyl)-N-[4- methoxyphenyl)ethyl]propenamide (58) (MeOD, 400 MHz)
Figure 3.48. DEPT NMR spectrum of 7 -(3 ,4 -dihydroxyphenyl)-N-[4- methoxyphenyl)ethyl]propenamide (58) in MeOD.
113
Figure 3.49. HSQC NMR spectrum of 7 -(3 ,4 -dihydroxyphenyl)-N-[4- methoxyphenyl)ethyl]propenamide (58) in MeOD.
Figure 3.50. HMBC NMR spectrum of 7 -(3 ,4 -dihydroxyphenyl)-N-[4- methoxyphenyl)ethyl]propenamide (58) in MeOD.
114
Figure 3.51. HMBC NMR spectrum of 7 -(3 ,4 -dihydroxyphenyl)-N-[4- methoxyphenyl)ethyl]propenamide (58) in MeOD (expanded scale).
Figure 3.52. COSY NMR spectrum of 7 -(3 ,4 -dihydroxyphenyl)-N-[4- methoxyphenyl)ethyl]propenamide (58) in MeOD.
115
8. Identification of maslinic acid (62)
(-)-Maslinic acid (62) was obtained as a white powder. A molecular formula of
+ C17H9NO3 was determined from the molecular ion peak at m/z 495.3433 [M+Na] (calcd for C17H9NO3Na, 495.3450) in the HRESIMS. The IR spectrum showed bands at 3400,
3100, and 1684 cm-1, indicative of hydroxy group, alkane C-H, and carbonyl stretching,
1 respectively. The H NMR spectrum showed signals for an olefinic proton at H 5.48 (1H,
brs, H-12; C 122.9); six singlets at H 0.96 (3H, s, 30-CH3; C 33.7), 1.00 (3H, s, 25-
CH3; C 17.3), 1.02 (3H, s, 29-CH3; C 24.2), 1.03 (3H, s, 26-CH3; C 17.9), 1.28 (3H, s,
27-CH3; C 26.6), 1.29 (3H, s, 24-CH3; C 29.8); and a carbonyl resonance at C 180.6 (C-
28). These NMR shifts along with a calculated degree of unsaturation of seven, were
characteristic of an olean-12-en-28-oic acid skeleton (Taniguchi et al., 2002). The
spectrum also exhibited signals for two oxygen-bearing methine protons at H 4.10 (1H, ddd, J = 11.0, 9.3, 4.1 Hz, H-2 ; C 69.0) and H 3.40 (1H, d, J = 9.3 Hz, H-3 ; C 84.3).
The coupling constant between H-2/H-3 was 9.3 Hz, suggestive of a 2 -OH, 3 -OH relative configuration of the diol, which was supported by NOESY correlation between
H-2 /CH3-24 and H-2 /CH3-25 . Since the Mosher ester procedure was considered
impractical for use for the assignments of the absolute configuration of the C-2/C-3 diol
of 61, induced circular dichroism (ICD) using a method developed by Snatzke and Frelek
was employed (Frelek et al., 1999; DiBari et al., 2001). As 1,2 diols are transparent in the
UV-vis region, the Cottonogenic reagent dimolybdenum tetracetate [Mo2(AcO)4] was
used to induce a CD curve through formation of a ligand-metal complex having a
chromophore. Formation of the Cottonogenic derivative resulted in a sterically
116 constrained diol with two diastereomorphous g+ and g- structural possibilities, dependent on the size of substituents around the vicinity of the complexed diol (DiBari et al., 2001).
Snatzke's rule states that band IV (~310 nm) has the same sign of the O-C-C-O dihedral angle in the favored conformation of the metal-ligand complex. In the ICD spectrum of
(-)-maslinic acid (62), the Cotton effect at 310 nm was negative, which corresponded to a negative dihedral angle of the O-C-C-O moiety. The absolute configuration of the C-2,
C-3 diol in compound 62 was assigned as 2R and 3R (Figures 3.64, 3.65). The physical and spectroscopic data were consistent with literature values for the known compound,
(-)-maslinic acid (Taniguchi et al., 2002; Tan et al., 2009; Yang et al., 2011).
The 1D- and 2D-NMR spectra obtained for this compound are presented in
Figures 3.53-3.63.
1 Figure 3.53. H NMR spectrum of (-)-maslinic acid (62) (pyridine-d5, 400 MHz)
117
1 Figure 3.54. H NMR spectrum of (-)-maslinic acid (62) (pyridine-d5, 400 MHz) (expanded scale)
13 Figure 3.55. C NMR spectrum of (-)-maslinic acid (62) in pyridine-d5
118
13 Figure 3.56. C NMR spectrum of (-)-maslinic acid (62) (pyridine-d5, 100 MHz) (expanded scale)
Figure 3.57. DEPT NMR spectrum of (-)-maslinic acid (62) (pyridine-d5)
119
Figure 3.58. DEPT NMR spectrum of (-)-maslinic acid (62) (pyridine-d5, expanded scale)
Figure 3.59. HSQC NMR spectrum of (-)-maslinic acid (62) in pyridine-d5
120
Figure 3.60. HSQC NMR spectrum of (-)-maslinic acid (62) (pyridine-d5, expanded scale)
Figure 3.61. HMBC NMR spectrum of (62) in pyridine-d5
121
Figure 3.62. COSY NMR spectrum of (-)-maslinic acid (62) (pyridine-d5)
Figure 3.63. NOESY NMR spectrum of (62) (pyridine-d5)
122
1.5 -0.5 255 280 305 330 355 380 405 430 455 480 -2.5 -4.5 -6.5 -8.5
-10.5CD [mdeg] -12.5 -14.5 -16.5 -18.5 Wavelength (nm) Figure 3.64. ECD spectrum of maslinic acid (62) in a DMSO solution (red). ECD spectrum maslinic acid (62) in a DMSO solution of Mo2(OAc)4 (blue).
4+ Figure 3.65. The O-C-C-O dihedral in the maslinic acid-[Mo2] complex.
123
9. Identification of palmitic acid (57)
Palmitic acid (57) was obtained as a white powder. A molecular formula of
+ C16H32O2 was determined from the molecular ion peak at m/z 279.2275 [M+Na] (calcd for C16H32O2Na, 279.2300) in the HRESIMS. The IR spectrum showed bands at 2954,
2916, 2849, and 1700 cm-1, indicative of alkane C-H and carbonyl stretching,
1 13 respectively. Diagnostic H and C NMR signals at H 1.41 (2H, m, H-4; C 30.4), H
1.82 (2H, m, H-3; C 26.3), and H 2.54 (2H, m, H-2; C 35.5) exhibited HMBC
correlations with the carboxylic acid moiety at C 176.7. The carboxylic acid hydroxy
group appeared at H 4.99 (brs). The remaining overlapped methylene signals appeared at
H 1.27 (22H, m, H-5 through H-15). The terminal methyl group was apparent at H 0.87
(3H, t, J = 7.9 Hz, H-16; C 14.9). The physical and spectroscopic data were consistent
with literature values for the known compound, palmitic acid (Liu et al., 2012; Marekov
et al., 2012).
The 1D-NMR spectra obtained for this compound are presented in Figures 3.66-
3.68.
124
1 Figure 3.66. H NMR spectrum of palmitic acid (57) (pyridine-d5, 400 MHz)
13 Figure 3.67. C NMR spectrum of palmitic acid (57) (pyridine-d5, 100 MHz)
125
13 Figure 3.68. C NMR spectrum of palmitic acid (57) (pyridine-d5, 100 MHz) (expanded scale)
10. Identification of (-)-epicatechin (34)
(-)-Epicatechin (34) was obtained as colorless needle crystals, mp 175-177 °C. A
molecular formula of C15H19NO4 was determined from the molecular ion peak at m/z
+ 313.0666 [M+Na] (calcd for C15H14O6Na, 313.0688) in the HRESIMS. The degree of unsaturation was calculated as nine from this mass, as seen in the structure of 34 with three rings and six double bonds. The IR spectrum showed bands at 3420 and 1652 cm-1, indicative of hydroxy group and olefinic C=C stretching, respectively. The 1H NMR signals at H 5.95 (1H, d, J = 2.3 Hz, H-8) and H 5.93 (1H, d, J = 2.3 Hz, H-6) were meta-coupled, as indicated by the magnitude of the coupling constant. Proton signals at
H 2.73 (1H, dd, J = 16.8, 2.7 Hz, H-4 ), 2.86 (1H, dd, J = 16.8, 4.5 Hz, H-4 ), 4.16 (1H, 126
m, H-3), and 4.79 (1H, brs, H-2) were observed and are typical of the flavan-3-ol class
(Zhang et al., 2012). A negative Cotton effect was observed at 240 nm and a negative
value at 280 nm, which is typical for a 2S,3S flavan-3-ol (Slade et al., 2005). A specific
25 rotation value of [ ]D -51 (c 1.0, CHCl3) supported a 2S, 3S configuration and was
comparable to reported literature values (Roy et al., 2009) (Figure 3.72). The remaining
ring B proton and carbon chemical shifts at H 6.79 (1H, dd, J = 8.3, 1.7 Hz, H-6 ; C
119.4), H 6.75 (1H, d, J = 8.0 Hz, H-5 ; C 115.9), and H 6.98 (1H, d, J = 2.1 Hz, H-2 ;
C 115.2) exhibited meta coupling between H-2 /H-6 and ortho coupling between H-
5 /H-6 . The arrangement of the ring B protons implied an ortho relationship of the
hydroxy groups at C-3 and C-4. The C-3 and C-4 hydroxy groups are considered
interchangeable as seen by the nearly identical chemical shifts of the oxygenated
aromatic carbons at C 145.61 (C-3 ) and C 145.78 (C-4 ). The physical and
spectroscopic data obtained were consistent with the cited literature for the known
compound, (-)-epicatechin (34) (Zhang et al., 2012).
The 1D-NMR spectra obtained for this compound are presented in Figures 3.69-
3.71.
127
Figure 3.69. 1H NMR spectrum of epicatechin (34) (MeOD, 300 MHz)
Figure 3.70. 13C NMR spectrum of epicatechin (34) (MeOD, 75 MHz)
128
Figure 3.71. 13C NMR spectrum of epicatechin (34) (MeOD, 75 MHz) (expanded scale)
Figure 3.72. CD curve for (-)-epicatechin (34)
129
11. Identification of daucosterol ( -sitosterol-3- -D-glycoside) (63)
Daucosterol (63) was obtained as a white powder. A molecular formula of
+ C35H60O2 was determined from the molecular ion peak at m/z 599.42832 [M+Na] (calcd for C35H60O2Na, 599.42821) in the HRESIMS. The degree of unsaturation was calculated
as six from this mass, as seen in the structure of 63 bearing five rings and one double
bond. The IR spectrum showed bands at 3377, 2868, and 1457 cm-1, indicative of
hydroxy group and alkane C-H group stretching, and methyl group bending, respectively.
The proton and carbon NMR spectra showed signals of an olefinic proton at H 5.36 (1H,
t, J = 4.4 Hz, H-6; C 122.4), and six singlets at H 0.67 (3H, s, 18-CH3; C 12.4), 0.87
(3H, brs, 26-CH3; C 19.6), 0.89 (3H, brs, 27-CH3; C 20.4), 0.91 (3H, brs, 29-CH3; C
12.6), 0.95 (3H, s, 19-CH3; C 19.4), 1.01 (3H, d, J = 6.4 Hz, 21-CH3; C 19.9). These
NMR shifts along with a calculated degree of unsaturation equal to six, were
characteristic of a 3-hydroxy-stigmastane skeleton possessing a 5,6 double bond (Zhang
et al., 2012). The proton and carbon spectra also exhibited signals for a glucose sugar
unit, inclusive of an anomeric proton signal at H 5.08 (1H, d, J = 7.6 Hz, H-1 ; C
103.1). The coupling constant between H-1 and H-2 (J = 7.6 Hz) was suggestive of a -
linked glucose moiety. The relative configuration of 63 was elucidated by polarimetry,
25 with a value of [ ]D -41.5 (c 0.1; MeOH) comparable to literature values for the known
compound, daucosterol (Behr and Leander, 1976; Wei et al. 2004; Suo and Yang, 2009).
The physical and spectroscopic data were consistent with literature values for daucosterol
(Behr and Leander, 1976; Wei et al. 2004; Suo and Yang, 2009).
130
The 1D-NMR spectra obtained for this compound are presented in Figures 3.73-
3.77.
1 Figure 3.73. H NMR spectrum of daucosterol (63) (pyridine-d5, 400 MHz)
1 Figure 3.74. H NMR spectrum of daucosterol (63) (pyridine-d5, 400 MHz) (expanded scale)
131
13 Figure 3.75. C NMR spectrum of daucosterol (63) (pyridine-d5, 100 MHz)
13 Figure 3.76. C NMR spectrum of daucosterol (63) (pyridine-d5, 100 MHz) (expanded scale, downfield region) 132
13 Figure 3.77. C NMR spectrum of daucosterol (63) (pyridine-d5, 100 MHz) (expanded scale, upfield region)
12. Identification of (-)-loliolide (65)
25 (-)-Loliolide (65) was obtained as a colorless solid, mp 101-102 °C and [ ]D
-105. A molecular formula of C11H16O3 was determined from the molecular ion peak at
+ m/z 219.0995 [M+Na] (calcd for C11H16O3Na, 219.0997) in the HRESIMS. The IR spectrum showed bands at 3436, 2947, 1724, and 1620 cm-1, indicative of olefinic hydroxy group, alkane C-H, olefinic C-H, and carbonyl stretching, respectively. The UV spectrum exhibited maxima at 295 nm and 260 nm suggesting conjugation in the
1 molecule. The H NMR spectrum showed germinal methyl signals at H 1.29 (3H, s, 1-
CH3 ) and 1.48 (3H, s, 1-CH3 ), that exhibited a COSY correlation, as well as HMBC
correlations to the quaternary carbon signal at C 36.3 (C-1). Methylene signals at H
133
[1.55 (1H, dd, J = 14.5, 3.6 Hz, H-2 ); 2.00 (1H, dt, J = 14.5, 2.5 Hz, H-2 ) C 47.6] and
[2.48 (1H, dt, J = 14.0, 2.5 Hz, H-4 ); 1.82 (1H, m, H-4 ); C 46.0] exhibited HMBC correlations to an oxygen-bearing methine at H 4.35 (1H, m, H-3 ). A low-pass J-filter set at 2-3 Hz was necessary to confirm the HMBC correlation between H-3 and the methylene at H-2/H-4. The low-pass filter HMBC experiment was supported by an 1H
13C H2BC experiment. The 1H 13C H2BC pulse sequence exclusively enhances two-bond correlations that can be absent in HMBC spectra due to vanishing two-bond coupling constants (Nyberg et al., 2005). Clear identification of two-bond correlations using the
H2BC experiment, when combined with the HSQC and HMBC spectra, facilitated
INADEQUATE-type correlations for compound 65 (Nyberg et al., 2005). The H2BC correlation between the signal at H 4.35 (1H, m, H-3 ) and the H-2/H-4 methylenes was readily observed. An olefinic proton resonance at H 5.71 (1H, s, H-7; C 113.3) showed
1 13 two-bond H C HMBC correlations to a lactone carbonyl moiety ( C 172.3), a quaternary carbon ( C 182.8), and the geminal dimethyl quaternary carbon at C 36.3
(Figure 3.84). When considered in combination with the four degrees of unsaturation evident for this compound, these NMR signals suggested a 6/5 norisoprenoid skeleton. A methyl group signal at H 1.80 (3H, s, CH3-5; C 27.4) was correlated to the quaternary carbon at C 182.8 as well as an oxygenated quaternary carbon at C 87.11. This suggested that methyl group at H 1.80 is located at the C-5 bridgehead position of compound 65. The configuration of (-)-loliolide was elucidated by measurement of
2 specific rotation, where a value of [ ]D -107.5 (c 0.1, MeOH) was comparable to literature values of this compound (Kuba et al., 2002; Pan et al., 2009). The configuration
134 was supported by a NOESY correlation between H 1.82 (H-4 ) and H 1.80 (CH3-5 ) suggesting these moieties were on the same face of the molecule (Figure 3.85). The physical and spectroscopic data were consistent with literature values for the known compound, (-)-loliolide (Valdes, 1986; Kuba et al., 2002; Pan et al., 2009).
The 1D- and 2D-NMR spectra obtained for this compound are presented in
Figures 3.78-3.85.
1 Figure 3.78. H NMR spectrum of (-)-loliolide (65) (CDCl3, 600 MHz)
13 Figure 3.79. C NMR spectrum of (-)-loliolide (65) (CDCl3, 150 MHz) 135
Figure 3.80. DEPT NMR spectrum of (-)-loliolide (65) (CDCl3)
Figure 3.81. HSQC NMR spectrum of (-)-loliolide (65) (CDCl3)
136
Figure 3.82. HMBC (CNST2, J filter = 10 Hz) NMR spectrum of (-)-loliolide (65) (CDCl3)
Figure 3.83. HMBC (CNST2, J filter = 3 Hz) NMR spectrum of (-)-loliolide (65) (CDCl3) 137
Figure 3.84. H2BC NMR spectrum of (-)-loliolide (65) (CDCl3)
Figure 3.85. NOESY NMR spectrum of (-)-loliolide (65) (CDCl3)
138
13. Synthesis of 2 -methyl-3 ,6 -(di-tert-butyldimethylsilyl)piperidine: overview of synthetic route
The synthesis of the 2R,3R,6R-trisubstituted piperidine analogue 72 (Scheme 3.1), was based on the enatioselective total synthesis of (2R,3R,6R)-N-methyl-6-(deca-1 ,3 ,5 - trienyl)-3-methoxy-2-methylpiperidine (Nakatani et al., 2006). Swern oxidation of the commercially available (R)-2-(Boc-amino)-1-propanol 75 and in-situ reaction with 3- butenylmagnesium bromide, afforded the syn-amino alcohol 66. The syn stereoselectivity is due to chelation-controlled addition of the Grignard reagent to the aldehyde (Veeresa and Datta, 1998). Protection of the hydroxy moiety (3-OH) as a tert-butyldimethylsilyl
(TBS) ether under basic conditions was achieved by treating with 2 equivalents of imidazole and 1.2 equivalents of tert-butyldimethylsilylchlorosilane (TBSCl) to give alkene 67. The alkene 67 was oxidized with polymer-bound OsO4 to the diol 68, followed by preferential TBS protection of the primary hydroxy group, controlled by equivalents of TBSCl added, to give 69. PDC oxidation of the secondary hydroxy group afforded 70.
An Ohfune-type deprotection of the N-tert-butoxycarbonyl (Boc) by treatment with excess tert-butyldimethylsilyl trifluoromethanesulfonate (TMS-triflate), 2,6-lutidine, triethylamine and methanol in dichloromethane resulted in cyclodehydration to form cyclic imine 71. Hydrogenation with a catalytic amount of Pd/C afforded the saturated piperidine 72. Since the Pd/C catalyst preferentially approaches the imine from the less hindered -face of the molecule, the piperidine 72 is syn (Nakatani et al., 2006) (Scheme
3.1).
139
Scheme 3.1 Synthesis of the 2 -methyl-3,6 -(di-tert-butyldimethylsilyl) piperidine analogue. Reagents and conditions. (i) Swern oxidn. then H2C=CH(CH2)2MgBr, 2 steps 61%; (ii) TBSCl, imidazole, DMF, 63%; (iii) OsO4, NMO, THF-acetone (1:1), quant. yield; (iv) TBSCl, imidazole, DMF, 65%; (v) PDC, CH2Cl2, 67%; (vi) 1) Me3SiOTf/2,6- lutidine 2) MeOH/Et3N in CH2Cl2 (vii) Pd/C, H2, MeOH, 2 steps 33%
14. Identification of (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-hydroxy-1-heptene
(66).
25 The Grignard amino alcohol 66 was obtained as a yellow oil, [ ]D +34 (c 0.1,
MeOH). A molecular formula of C12H23NO3 was determined from the molecular ion peak
+ at m/z 252.1570 [M + Na] (calcd for C12H23NO3, 252.1576) in the HRESIMS, which
indicated the presence of a single nitrogen atom in the molecule. The IR spectrum
showed absorption bands at 3300-3500, 2900, and 1650 cm-1, suggestive of hydroxy
(-OH), amine (-N-H), alkane C-H, and carbonyl group stretching, respectively. The 1H
NMR spectrum (300 MHz, CDCl3) (Figure 3.86) included a methyl group [ H 1.14 (3H, d, J = 6.69 Hz, 2-CH3)], two methylene groups [ H 1.45 (2H, m, 3-H2), H 2.17 (2H, m, 4-
140
H2), an oxygen-bearing methine group [ H 4.85 (1H, m, H-5)], Boc tert-butyl methyl groups at H 1.41 (9H, s, Boc), and olefinic signals at H 4.83 (1H, dd, J = 17.1, 8.7 Hz,
H-1a) and 5.05 (1H, dd, J = 14.5, 8.7 Hz, H-1b) and 5.70 (1H, m, H-2). The physical and
spectroscopic data were consistent with literature values for (5R,6R)-6-[(tert)-
butoxycarbonyl)amino]-5-hydroxy-1-heptene (66) (Kumar and Datta, 1999; Nakatani et
al., 2006). The amino alcohol 66, was assigned structurally, therefore, as (5R,6R)-6-
[(tert)-butoxycarbonyl)amino]-5-hydroxy-1-heptene.
Figure 3.86. 1H NMR spectrum of (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-hydroxy- 1-heptene (66) (CDCl3, 300 MHz)
141
15. Identification of (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-(tert-
butyldimethylsilyl)-1-heptene (67).
The tert-butyl dimethysilyl (TBS) protected amino alcohol 67 was obtained as a
25 colorless oil, [ ]D +38 (c 0.1, MeOH). A molecular formula of C18H37NO3Si was determined from the molecular ion peak at m/z 366.2371 [M + Na]+ (calcd for
C18H37NO3SiNa, 366.2440) in the HRESIMS, which indicated the presence of a single
nitrogen atom in the molecule. The IR spectrum showed absorption bands at 3450, 2930,
and 1716 cm-1, indicative of amine (-N-H), alkane C-H, and carbonyl group stretching,
1 respectively. The H NMR spectrum (300 MHz, CDCl3) (Figure 3.87) was closely
identified with 67 but included additional resonances for a tert-butyl dimethysilyl methyl
group at H 0.02 (6H, s, TBS-CH3), and H 0.85 (9H, s, TBS-tert-butyl), indicating that
the 5-OH secondary hydroxy moiety in 67 was successfully protected. The amino alcohol
67 was assigned structurally as (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-(tert-
butyldimethylsilyl)-1-heptene.
142
Figure 3.87. 1H NMR spectrum of (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-(tert- butyldimethylsilyl)-1-heptene (67) (CDCl3, 300 MHz)
16. Identification of (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-(tert- butyldimethylsilyl)-1,2-diol (68).
25 The diol 68 was obtained as a colorless oil, [ ]D +45 (c 0.1, MeOH). A molecular formula of C18H39NO5Si was determined from the molecular ion peak at m/z
+ 400.2466 [M + Na] (calcd for C18H39NO5SiNa, 400.2495) in the HRESIMS, which indicated the presence of a single nitrogen atom in the molecule. The IR spectrum showed absorption bands at 3500, 3447, 2930, 1716 cm-1, consistent with a free hydroxy group, amine (-N-H) moiety, alkane C-H, and carbonyl group stretching, respectively.
1 The H NMR spectrum (300 MHz, CDCl3) (Figure 3.88) was closely identified with 67 but included additional resonances for an oxygen-bearing methine proton at H 4.68 (1H,
143 bd, J = 9.24, H-2 ), and oxygen-bearing methylene protons at H 3.38-3.73 (2H, m, H-1a,
H-1b), indicating the formation of a 1,2-diol functionality. The relative configuration of the diol was confirmed by comparing optical rotation values with the literature and the
NOESY correlation (Figure 3.89) between H-2 /H-6 . The physical and spectroscopic
data were consistent with literature values for (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-
5-(tert-butyldimethylsilyl)-1,2-diol (68) (Nakatani et al., 2006). The diol 68 was assigned structurally, therefore, as (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-(tert- butyldimethylsilyl)-1,2-diol.
Figure 3.88. 1H NMR spectrum of (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-(tert- butyldimethylsilyl)-1,2-diol (68) (CDCl3, 300 MHz)
144
Figure 3.89. NOESY NMR spectrum of (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5- (tert-butyldimethylsilyl)-1,2-diol (68) (CDCl3)
17. Identification of (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-(tert- butyldimethylsilyl)-1-(tert- butyldimethylsilyl)-2-ol (69).
25 The TBS protected terminal alcohol 69 was obtained as a colorless oil, [ ]D +28
(c 0.1, MeOH). A molecular formula of C24H53NO5Si was determined from the molecular
+ ion peak at m/z 514.3342 [M + Na] (calcd for C24H53NO5Si2Na, 514.3360) in the
HRESIMS, which indicated the presence of a single nitrogen atom in the molecule. The
IR spectrum showed absorption bands at 3500, 3447, 2930, and 1716 cm-1 indicative of a free hydroxy group, amine (-N-H) moiety, alkane C-H, and carbonyl group stretching,
1 respectively. The H NMR spectrum (300 MHz, CDCl3) (Figure 3.90) was comparable to that of 68 but included an additional overlapped resonance for a tert-butyl dimethysilyl methyl group at H 0.08 (12H, s, TBS-CH3), and H 0.92 (18H, s, TBS-tert-butyl),
145 indicating that the 1-OH primary hydroxy moiety in 68 was successfully TBS protected.
Intermediate 69 was assigned structurally as (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-
(tert-butyldimethylsilyl)-1-(tert- butyldimethylsilyl)-2-ol.
Figure 3.90. 1H NMR spectrum of (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-(tert- butyldimethylsilyl)-1-(tert- butyldimethylsilyl)-2-ol (69) (CDCl3, 300 MHz)
18. Identification of (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-(tert-
butyldimethylsilyl)-1-(tert-butyldimethylsilyl)-2-heptanone (70).
The PDC oxidation derived secondary ketone 70 was obtained as a colorless oil,
25 [ ]D +25 (c 0.1, MeOH). A molecular formula of C24H51NO5Si2 was determined from
+ the molecular ion peak at m/z 512.3188 [M + Na] (calcd for C24H51NO5Si2Na,
512.3204) in the HRESIMS, consistent with the presence of a single nitrogen atom in the
146 molecule. The IR spectrum showed absorption bands at 3451, 2955, and 1716 cm-1 indicative of amine (N-H), alkane C-H, and carbonyl group stretching, respectively. The
1 H NMR spectrum (300 MHz, CDCl3) (Figure 3.91) was characterized by a diagnostic resonance at at H 4.19 (2H, s, H-1a, H-1b), indicating that the C-2 secondary hydroxy group in 70 was reduced to the ketone, resulting in a pair of magnetically equivalent H-1 methylene protons. Key COSY correlations (Figure 3.92) included the methylene resonances at H 2.56 (2H, t, J = 5.7 Hz, H-3a, H-3b) and 1.71 (2H, m, H-4a, H-4b); H-4 and 4.63 (1H, bd, J = 6.36 Hz, H-5); and H-5 and the signal at H 3.72 (1H, m, H-6). The physical and spectroscopic data were consistent with literature values for (5R,6R)-6-
[(tert)-butoxycarbonyl)amino]-5-(tert-butyldimethylsilyl)-1-(tert-butyldimethylsilyl)-2- heptanone (12) (70) (Nakatani et al., 2006). The ketone 70 was assigned structurally, therefore, as (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-(tert-butyldimethylsilyl)-1-(tert- butyldimethylsilyl)-2-heptanone.
147
Figure 3.91. 1H NMR spectrum of (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-(tert- butyldimethylsilyl)-1-(tert- butyldimethylsilyl)-2-heptanone (70) (CDCl3, 300 MHz)
Figure 3.92. COSY NMR spectrum (5R,6R)-6-[(tert)-butoxycarbonyl)amino]-5-(tert- butyldimethylsilyl)-1-(tert- butyldimethylsilyl)-2-heptanone (70) (CDCl3) 148
19. Identification of of (2R,3R,6R)-3-[(tert)-butyldimethylsilyl)]-2-methyl-6-(tert-
butyldimethylsilyl)piperidine (72).
The 2R,3R,6R-trisubstituted piperidine analogue 72 was obtained as a yellow oil,
25 [ ]D -41.7 (c 0.1, MeOH). A molecular formula of C19H43NO2Si2 was determined from
+ the molecular ion peak at m/z 396.2716 [M + Na] (calcd for C19H43NO2Si2Na, 396.2730)
in the HRESIMS, which indicated the presence of a single nitrogen atom in the molecule.
The IR spectrum showed absorption bands at 3200, 3150, and 2955 cm-1 indicative of a
secondary amine N-H and olefinic C-H stretching, respectively. The 1H and 13C NMR
spectra (300 MHz, CDCl3) included a methyl group at [ H 1.01 (3H, d, J = 6.57 Hz, 2 -
CH3); C 19.4], two methylene groups at [ H 1.56 (1H, m, H-4 ), H 1.86 (1H, m, H-4 ),
H 1.28 (1H, m, H-5 ), H 1.73 (1H, m, H-5 ); C 32.4 (C-4), C 21.9 (C-5)], one oxygen-
bearing methine group [ H 3.53 (1H, m, H-3); C 68.0 (C-3)], two nitrogen-bearing
methine groups [ H 2.48 (1H, dq, J = 12.6, 6.12 Hz, H-2), 2.57 (1H, m, H-6); C 19.4 (C-
2), C 57.3 (C-6)], silyl dimethyl groups at H 0.03 (12H, bs, TBS-CH3), and tert-butyl
methyl groups at H 0.89 (12H, s, TBS). The oxygen-bearing methine group at H 3.53
(C-3) showed a COSY correlation with the nitrogen-bearing methine group at H 2.48 (C-
2). The methylene signals H 1.28 and H 1.73, corresponding to the diastereotopic C-5
protons, showed COSY correlations with the nitrogen-bearing methine group at H 2.57
1 13 (C-6). A heteronuclear H C HMBC three-bond correlation between 2 -CH3 ( H 1.01)
and C-3 ( C 68.0), and a two-bond correlation between both diastereotopic C-5 protons
and the nitrogen-bearing methine group at H 2.57 (C-6), were observed. Taken together,
the chemical shift values and connectivity patterns from the COSY and HMBC spectra 149 indicated the presence of a 2-methyl-3,6-di-tert-butyldimethylsilyl piperidine ring. The syn stereochemistry of 72 was confirmed from the NOESY spectrum and analysis of the
1 H NMR coupling patterns. A NOESY correlation between the signals at H 2.68 (1H, dq,
J = 12.6, 6.12 Hz, H-2), H 3.53 (1H, m, H-3), and H 2.57 (1H, m, H-6) indicated these substituents to be on the same face of the piperidine ring. The optical rotation value of
25 25 [ ]D -41.7 (c 0.1, MeOH) is comparable to the syn mono-pivalate intermediate [ ]D -
19.2 (c 1.0, MeOH) reported in the synthesis of (2R,3R,6R)-N-methyl-6-(deca-1 ,3 ,5 - trienyl)-3-methoxy-2-methylpiperidine (Nakatani et al., 2006). The total synthesis of 72 was achieved in 6% overall yield in eight steps from 75. The synthetic piperidine 72 was assigned structurally as 2 -methyl-3,6 -(di-tert-butyldimethylsilyl) piperidine.
The 1D- and 2D-NMR spectra obtained for this compound are presented in
Figures 3.93-3.99.
Figure 3.93. 1H NMR spectrum of (2R,3R,6R)-3-[(tert)-butyldimethylsilyl)]-2-methyl-6-(tert- butyldimethylsilyl)piperidine (72) (CDCl3, 300 MHz)
150
Figure 3.94 13C NMR spectrum of (2R,3R,6R)-3-[(tert)-butyldimethylsilyl)]-2-methyl-6-(tert- butyldimethylsilyl)piperidine (72) (CDCl3, 75 MHz)
Figure 3.95. DEPT NMR spectrum of (2R,3R,6R)-3-[(tert)-butyldimethylsilyl)]-2- methyl-6-(tert-butyldimethylsilyl)piperidine (72) (CDCl3)
151
Figure 3.96. HSQC NMR spectrum of (2R,3R,6R)-3-[(tert)-butyldimethylsilyl)]-2- methyl-6-(tert-butyldimethylsilyl)piperidine (72) (CDCl3)
Figure 3.97. HMBC NMR spectrum of (2R,3R,6R)-3-[(tert)-butyldimethylsilyl)]-2- methyl-6-(tert-butyldimethylsilyl)piperidine (72) (CDCl3) 152
Figure 3.98. COSY NMR spectrum of (2R,3R,6R)-3-[(tert)-butyldimethylsilyl)]-2- methyl-6-(tert-butyldimethylsilyl)piperidine (72) (CDCl3)
Figure 3.99. NOESY NMR spectrum of (2R,3R,6R)-3-[(tert)-butyldimethylsilyl)]-2- methyl-6-(tert-butyldimethylsilyl)piperidine (72) (CDCl3)
153
20. Biological test data obtained
All the isolated compounds from active fractions obtained from the stem bark,
branches, and leaves of Microcos paniculata were evaluated for their cytotoxicity against
HT-29 human colon cancer cells. Microgrewiapine A (56) was found to be the most
active pure compound in inhibiting the proliferation of HT-29 cancer cells, with an IC50 value of 6.8 µM. Screening of 56 against CCD-112CoN normal colon cells resulted in an
IC50 value of 30.4 µM. Therefore, the selectivity ratio of 56 for cancerous colon cells
versus normal colon cells was found to be approximately 4.0 (Table 3.4).
Microgrewiapine 3-acetate, compounds 58-61, 64 and 72 showed cytotoxicity values of
greater than 10 µM when evaluated against the HT-29 cancer cell line, and were therefore
deemed inactive (Table 3.4).
Structural similarities between 56 and 1-(3-phenylpropyl)piperidine-3-yl)methyl
[1,1 -biphenyl]-2-carboxylate (KAB-18), which was previously identified as a novel
ligand of neuronal nicotinic receptors (nAChRs), prompted an examination of the effects
of the compounds isolated in this dissertation work on nAChRs (González-Cestari et al.,
2009). In particular, the phenyl rings, the three-carbon aliphatic chain, and the piperidine
ring in KAB-18 are comparable to the triene side-chain, aliphatic tail, and piperidinol
ring found in 56. The effects of the nitrogen containing isolates from M. paniculata on
human nAChR activity were tested using a functional calcium accumulation assay with
HEKtsA201 cells stably expressing either human h 4 2 or h 3 4 nAChRs. When
assayed at a single concentration (10 µM), the natural products (56, 58-61, 64), the semi-
synthetic acetate analogue of 56 (microgrewiapine A 3-acetate), and the synthesized 154
piperidine analogue 72 inhibited the epibatidine-stimulated calcium accumulation of
h 4 2 or h 3 4 nAChRs (Figures 3.100- 3.102; Table 3.6). This supports their
determination as antagonists of human nAChRs. Specifically, microgrewiapine A (56)
exhibited approximately 60% and 70% inhibition of h 4 2 and h 3 4 nAChR activity,
respectively. In comparison, the acetate of 56 inhibited approximately 40% of the h 4 2
nAChR response (the acetate was not tested against h 3 4 nAChR). Analogues
incorporating an N-oxide moiety (60 and 61) also caused ~80% inhibition for both the
h 4 2 and h 3 4 nAChRs. Microcosamine A (64) inhibited 53.7% and 59.0% of the
h 3 4 and h 4 2 activity, respectively. Compounds 58 and 59 produced only weak
inhibition of activity for both the h 3 4 and h 4 2 nAChRs (less than 20% inhibition)
(Figure 3.100; Table 3.5).
Calcium accumulation assays were also performed to obtain dose-response IC50 curves. Experiments were performed in HEKts201 cells stably expressing either human h 4 2 or h 3 4 nAChRs (González-Cestari et al., 2009; Henderson et al., 2010).
Compounds 56, 58-61, 64, the acetate of 56, and 72, inhibited epibatidine-stimulated
calcium accumulation on h 4 2 nAChRs with IC50 values of 4.6 (3.2-6.7) µM, 81.0
(64.3-102.0), 10.9 (4.9-24.5), 2.6 (2.0-3.4) µM, 4.0 (1.4-11.1) µM, 89.8 (70.8-114.0),
10.6 (8.0-14.1) µM, and 47.4 (32.9-67.7) µM, respectively (Figure 3.100-3.102; Table
3.6). Inhibitory activity was also found on h 3 4 nAChRs with IC50 values for
compounds 56, 58, 60, and 61 of 8.3 (7.1-9.7) µM, 51.9 (37.6-71.4) µM, 2.9 (1.9-4.6)
µM, and 2.4 (1.1-5.4) µM, respectively. Compound 64 showed an IC50 value of >100
µM on h 3 4 nAChRs. Compounds 58 and 59 were shown to be more than ten-fold less
155 potent than all other compounds tested against either h 4 2 or h 3 4 nAChRs. No selectivity was found among these compounds for the nAChR subtypes (Figure 3.101;
Table 3.6). The potencies of these natural products are similar to potencies of other established nAChR antagonists including dihydro- -erythroidine, D-tubocurarine, tetracaine, bupropion, mecamylamine, and other KAB-18-like analogues (González-
Cestari et al., 2009; Henderson et al., 2010).
The competitive or non-competitive nature of the inhibitory activity was investigated by determining the effects of increasing concentrations of epibatidine in the absence and presence of microgrewiapine A (56). The effect of epibatidine in the presence of 56 was not surmountable with increasing concentrations of epibatidine
(maximum effect of 41.2 ± 3.3 %), compared to this agonist alone (95.1 ± 5.6 %) (Figure
3.102). This supports the classification of microgrewiapine A (56) as a noncompetitive nAChR antagonist with potential allosteric actions at the h 4 2 receptor subtype.
The presumed structural features of microgrewiapine A (56) that contribute to cytotoxicity and nAChR antagonism include hydrogen bond donors and acceptors, the configuration of the substituents in the piperidine ring, and the alkene side chain. To support this hypothesis, the total synthesis of (2R,3R,6R)-3-[(tert)-butyldimethylsilyl)]-2- methyl-6-(tert-butyldimethylsilyl)piperidine was accomplished in 6% overall yield, in eight steps from the N-Boc amino alcohol 75. The synthesis was based on the total synthesis of (2R,3R,6R)-N-methyl-6-(deca-1 ,3 ,5 -trienyl)-3-methoxy-2-methylpiperidine
(51) (Nakatani et al., 2006). The tert-butyldimethylsilyl (TBS) protected synthetic product (72) lacks hydrogen bond donors and acceptors (excluding the N-H moiety), is of
156 a different absolute configuration when compared to microgrewiapine A (56), and lacks the presence of an alkene sidechain. Any one of these structural changes from 56 may contribute to the diminished cytotoxicity and nAChR activity of the synthetic analogue
72. The C-3 acetate of microgrewiapine A, showed diminished activity, suggesting that a hydrogen bond donor and acceptor at this position contributes to both cytotoxicity and nAChR activity (Figure 3.100-3.101; Table 3.6). Compounds bearing a N-methyl moiety
(56, 60-61) exhibited higher nAChR potency than compounds with a N-H moiety (64). In addition, the commercially available analogues, 1,3-dimethylpiperidine (73) and piperidin-3-ol (74), both racemic, and without an alkene side chain were inactive (Figure
3.100; Table 3.6). The presence of the N-oxide moiety in microgrewiapine B (60) and microgrewipine C (61) diminished cytotoxicity, but slightly improved nAChR activity when compared to microgrewiapine A (56). Changing the structural class from a piperidine diminishes cytotoxicity and nAChR acitivty, as seen in the diminished activities of the amide derivative (58) and the isoquinoline, liriodenine (59).
157
140 h 3 4 h 4 2 120
100
80
60
40 (% Epibatidine) 20 Intensity Fluorescence 0 56 60 61 64 58 59 KAB-18 Compound
Figure 3.100. Effects of isolated and synthetic antagonists on recombinant nAChRs. An inhibition assay for each compound was performed using HEK tsA201 cells stably expressing h 3 4 and h 4 2 nAChRs. Cells were loaded with Calcium 5 NW dye and stimulated with 1 µM epibatidine in the presence of a single concentration of each test compound (10 µM). Results are expressed as the percentage of the control, epibatidine-stimulated peak fluorescence level. Values represent the means ± SD of three to five experiments performed in triplicate.
120 120 56 56 60 100 56 3-acetate 60 100 61 61 64 80 64 80 58 58 59 59 KAB-18 60 KAB-18 60
40 40 (% (% Epibatidine) (% (% Epibatidine) 20 20 Fluorescence Intensity Fluorescence Fluorescence Intensity Fluorescence 0 h 4 2 0 h 3 4 -8 -7 -6 -5 -4 -3 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 10 10 10 10 10 10 [Compound] (M) [Compound] (M)
Figure 3.101. Concentration-response effects of isolated and synthetic antagonists on recombinant nAChRs. Concentration-response studies of compounds on epibatidine stimulated calcium accumulation in HEK tsA201 stably expressing h 4 2 and h 3 4 nAChRs. Cells were loaded with Calcium 5 NW dye and stimulated with 1 µM epibatidine in the presence of increasing concentrations of the compounds tested. Results are expressed as percentage of control, epibatidine-stimulated peak fluorescence levels. Values represent the mean ± SD of three to five experiments performed in triplicate. 158
compound 56
Figure 3.102. Concentration-response effects of epibatidine in the absence or presence of microgrewiapine A (56). The concentration-response effects of epibatidine were investigated in the absence ( ) or presence ( ) of 10 µM compound 56 by using HEK tsA201 cells stably expressing h 4 2 nAChRs. Values represent means ± SD (n = 3) performed in triplicate.
D. Conclusions
In this dissertation work, the leaves, stem bark and branches of M. paniculata were collected in Vietnam in December 2008, through a benefit sharing agreement between the University of Illiniois at Chicago and the Vietnamese Academy of Science and Technology. This work was conducted as part of a program project funded through
The Ohio State University. Bioactivity-guided isolation using HT-29 human colon cancer cells resulted in the purification of eleven compounds, in particular, the cytotoxic microgrewiapine A (56). The compounds were evaluated against the h 4 2 and h 3 4 nAChR subtypes. Certain of the isolated natural products in this work exhibited antagonistic activity comparable to the established nAChR antagonists D-tubocurarine and mecamylamine. Significant to this study is that the cancer cell cytotoxicity and
159 nAChR activity shown by the isolated compounds together represent new bioactivities for compounds isolated from the genus Microcos.
Growth-inhibitory effects of piperidine alkaloids from M. paniculata stem bark on the second instar larvae of the mosquito Aedes aegypti, showed that piperidine alkaloids with similar chemical structures to those isolated in this thesis work, have insecticidal activity (Bandara et al., 2000). Despite the toxicity shown towards mosquito larvae, the leaves of M. paniculata are likely less toxic to humans than other plant parts, due to the low yield of microcosamine A (64) obtained from the leaves (0.005%), when compared to the yields of microgrewiapine A (56) from the stem bark (0.08%), and microgrewiapine B (60) (0.03%) and microgrewiapine C (61) (0.02%), from the branches. In line with the finding of lower yields of potentially toxic piperidine alkaloids in the leaves, this plant part has ethnomedicinal use as an herbal tea for the treatment of cold, enteritis, and skin rash in southern mainland China (Feng et al., 2008).
Given the evidence implicating nAChRs in the development and progression of cancer, the cytotoxicity observed for microgrewiapine A (56) against HT-29 human colon cancer cells in this investigation may be influenced by nAChR modulation. There is growing evidence implicating nAChRs in various aspects of cancer development and progression. nAChRs can influence the function, growth, and survival of cancer cells by regulation of a number of neurotransmitters and growth, angiogenic and neurotrophic factors [(e.g., dopamine, glutamate, -amino-butyric acid (GABA), serotonin, BDNF,
VEGF, HGF, TGF- , TGF- , and PDGF)] (Paleari et al., 2008; Schuller, 2009). In
160
addition, nAChR activation and the resulting increases in intracellular Ca2+ concentrations lead to stimulation of a number of signaling pathways mediating cell proliferation (e.g., Src kinase cascade, PI3-Akt pathway, ERK/MAP kinase cascade, NF-
B pathway, and cyclic AMP signaling cascade). Through activation of anti-apoptotic proteins and induction of NF- B, nAChRs also directly influence cell survival (Dasgupta and Chellappan, 2006; Paleari et al., 2008). Although the in vitro cytotoxicity of 56 may
be influenced by nAChR modulation, exploration of this mechanism is outside the scope
of this investigation, but presents a worthy future direction.
161
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Appendix A: Cell Culture and Cytotoxicity Assays
HT-29 Cytotoxicity Assay. Human colon cancer cells (HT-29) were obtained from American Type Collection (ATCC catalog no. HTB-38). Cells were cultured in
MEME medium (Hyclone, Logan, UT) supplemented with streptomycin (100 µg/mL), penicillin (100 units/mL), amphotericin B (Fungizone, 0.25 µg/mL), and 10% fetal bovine serum (FBS) and incubated in a humidified incubator with an atmosphere of 95% air and 5% CO2 at 37 °C. Cells were trypsinized and split for subculture when they reached near-confluent state (five days or later). Upon reaching about 60-70% confluence, the medium was changed and the cells were used for test procedures one day later. The harvested cells, after appropriate dilutions, were seeded in 96- well (9500 cells/190 µL) plates using complete medium and treated with the test compounds (10 µL/well in triplicate) at various concentrations. Test samples were initially dissolved in DMSO and then diluted 10-fold with H2O. Serial dilutions were performed using 10% DMSO as the solvent. For the control groups, 10 µL of 10% DMSO was also added to each well. The plates were incubated for three days at 37 °C in 5% CO2. On the third day, the cells were
fixed to the plates by the addition of 100 µL of cold 20% trichloroacetic acid and incubated at 4 °C for 30 min. The plates were washed three times with tap water and dried overnight. The fixed cells were dyed with sulforhodamine B (SRB, an anionic protein stain) solution at 0.4% (w/v) in 1% acetic acid and incubated at room temperature
174
for 30 min. The plates were washed three times with 1% acetic acid and allowed to air-
dry. The bound SRB stain was then solubilized with 10 mM unbuffered Tris base (pH 10,
200 µL/well). The plates were placed on a shaker for 5 min, and the absorbance was read
at 515 nm using a Bio-Tek µQuant microplate reader. The ED50 values of test samples
with serial dilutions were calculated using nonliner regression analysis (Table
Curve2Dv4; AISN Software, Inc., Mapleton, OR). Paclitaxel was used a positive control
and exhibited an IC50 value of 0.006 µM.
Normal Colon Cell Screening. Microgrewiapine A (56), with an IC50 value less than 10 µM against HT-29 cells was further tested against the CCD-112CoN normal colon cell line. For this assay, CCD-112CoN non-cancerous human colon cells (ATCC,
CRL-1541) were cultured in MEME medium (Hyclone, Logan, UT), supplemented with
10% FBS, PSF, 1.0 mM sodium pyruvate, 0.1 mM non-essential amino acid, and 1.5 g/L sodium bicarbonate, and placed in a humidified incubator with an atmosphere of 95% air and 5% CO2 at 37 °C. Cells were trypsinized and split for subculture when they reached
near-confluent state (five days or later). Upon reaching about 70% confluence, the
medium was changed and the cells were used for the test procedure one day later. The
harvested cells, after appropriate dilution, were seeded per well in 96-well CCD-112CoN
normal colon cells (7,600 cells/190 µL) plates, using complete medium and were treated
with the test samples (10 µL/well in triplicate) at various concentrations. Testing was
performed using 10% DMSO as the solvent. Then, 10 µL of 10% DMSO were also added
to the control wells. After incubation for 3 days at 37 °C in 5% CO2, the cells were fixed
to the plates by the addition of 100 µL of cold 20% trichloroacetic acid (TCA) and
175 incubated at 4 °C for 30 min. Next, the plates were washed three times with tap water and dried overnight. The fixed cells were stained for 30 min at room temperature with 0.4%
(w/v) sulforhodamine B (SRB), an anionic protein stain in 1% acetic acid, and rinsed three times with 1% acetic acid to remove unbound dye and allowed to dry. The bound dye was then solubilized with 10 mM unbuffered Tris base (pH 10, 200 µL/well) for 5 min on a shaker. Absorbance at 515 nm was measured with a Bio-Tek µQuant microplate reader. The IC50 values of test samples in serial dilutions was calculated using non-linear regression analysis (Table curve2Dv4; AISN Software, Inc., Mapleton OR). Paclitaxel was used as a positive control and exhibited an IC50 value of 23.0 µM against CCD-
112CoN normal colon cells.
176
Appendix B: Calcium Accumulation Assays
Calcium accumulation assays were performed with HEK ts201 cells stably expressing either human 3 4 nAChRs (h 3 4 nAChRs) or human 4 2 nAChRs (h 2 nAChRs) (obtained from Professor Jon Lindstrom, University of Pennsylvania,
Philadelphia, PA). Cells were plated at a density of 2.0 to 2.3 X 105 cells per well in poly-
L-ornithine coated 96-well culture plates 24 h prior to the assay. On the day of experiments, cells were washed with 100 µL of HEPES-buffered Krebs (HBK) solution and incubated with 40 µL of HBK and 40 µL of Calcium 5 NW dye (Molecular Devices,
Sunnyvale, CA) (1 h, room temperature). Changes in intracellular calcium levels resulting from nAChR activation were then measured simultaneously during and after the course of drug treatment period using a fluid handling integrated fluorescence plate reader (FlexStationII, Molecular Devices, Sunnyvale, CA). In order to evaluate the antagonistic effects of compounds, cells were treated with the compounds at the first addition (20 sec), and with 1 µM of the nAChR agonist epibatidine (Thermo Fisher
Scientific, Pittsburgh, PA) at the second addition (60 sec) in the continued presence of the compounds. For the quantification of compound antagonistic effects, a control-agonist treated group and a control-sham group were included. For the control-agonist group,
HBK (40 µL) and 1 µM of epibatidine solution (40 µL) were added at the first and second treatment period, respectively. The control-sham group was treated with HBK (40
177
µL) at the first and second treatment period, respectively. The changes in fluorescence were measured continuously for 120 sec from the bottom of the well at excitation of 485 nm and emission of 525 nm at ca. 1.5 sec intervals. Functional responses were quantified by first calculating the net fluorescence (the difference between control-agonist treated group and control-sham group) and results were expressed as a percentage of control- agonist group (1 µM epibatidine). All functional data were calculated from the number of observations performed in triplicate. For pharmacological evaluation, compounds were initially prepared with 100% DMSO (0.01 M stocks). Further dilutions of compounds were made in HBK buffer ( 100 µM).
178