I. ISOLATION AND CHARACTERIZATION OF BIOACTIVE COMPOUNDS FROM
SURINAME AND MADAGASCAR FLORA. II. A SYNTHETIC APPROACH TO
LUCILACTAENE
Eba Adou
Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in
partial fulfillment of the requirement for the degree of
Doctor of Philosophy
In
Chemistry
Dr. David G. I. Kingston, Chairman
Dr. James Tanko
Dr. Felicia Etzkorn
Dr. Timothy E. Long
Dr. Paul Deck
November 28, 2005
Blacksburg, Virginia
Keywords: Anticancer agents, Cytotoxicity assay, Indole alkaloids, Diterpenoids, Cardenolide glycosides, Physalins, Cucurbitacins, Cell cycle, Cell cycle inhibitor, p53 Tumor suppressor
Gene, Lucilactaene
Copyright 2005, Eba Adou ISOLATION AND CHARACTERIZATION OF BIOACTIVE COMPOUNDS FROM
SURINAME AND MADAGASCAR FLORA AND A SYNTHETIC APPROACH TO
LUCILACTAENE
ABSTRACT
Eba Adou
As part of an International Cooperative Biodiversity Group (ICBG), extracts of plants from Suriname and Madagascar were bioassayed for cytotoxicity and antimalarial activity. Six cytotoxic extracts and one potential antimalarial were selected for fractionation, and yielded a number of bioactive compounds which were characterized by spectroscopy methods.
Craspidospermum verticillatum (Apocynaceae) yielded four known indole alkoids. Casimirella sp (Icacinaceae) gave three new and five known diterpenoids. Pentopetia androsaemifolia
(Apocynaceae) afforded one new and three known cardenolide glycosides. Physalis angulata
(Solanaceae) yielded seven known physalins. Roupellina boivinnii (Apocynaceae) yielded four known and three new cardenolide glycosides, and three known cucurbitacins were isolated from
Octolepis aff. dioica (Thymelaeaceae).
In addition to these structural studies, a synthetic approach to lucilactaene, a cell cycle inhibitor was developed.
ACKNOWLEDGMENTS
I dedicate this to my family in Côte d’Ivoire (Ivory Coast) for giving me the opportunity to come to the U.S.A. I have been fortunate to come from a family who gave me all the opportunities and love to succeed in life.
I would like to thank my beloved wife, Dr. Nan Chi Wan, for her emotional support and her unconditional love.
I would like to thank Dr. David G.I. Kingston for his kindness, tolerance and support he has shown during my time in his research group. I would also like to thank my committee members: Dr. James Tanko, Dr. Felicia Etzkorn, Dr. Timothy E. Long and Dr. Paul Deck.
I would also like to thank Dr. Cao Shugeng for his help and friendship, without forgetting his wife, Xiaohua Wu and his son, Longji for their kindness.
Additional thanks to Jennifer Schilling and Tom Glass for technical assistance with bioassays and NMR spectroscopy.
III
TABLE OF CONTENTS
PART I
I. GENERAL INTRODUCTION 1
1.1 Cancer 1
1.2 Natural Products in Drug Discovery 1
1.3 Natural Products as Anticancer Drugs 2
1.4 Biodiversity loss and the ICBG Program 5
1.5 Bioassays in the discovery of antitumor agents 6
1.5.1 Role of assays 6
1.5.2 Antitumor assays 7
1.6 A2780 Cytotoxicity Assay 9
II CRASPIDOSPERMUM VERTICILLATUM (APOCYNACEAE) 11
2.1 Introduction 11
2.2 Results and Discussion 11
2.3 Experimental Section 15
2.3.1 General Experimental Procedures 15
2.3.2 Cytotoxicity Bioassays 16
2.3.3 Plant Material 17
2.3.4 Extraction and Isolation 17
III. CASIMIRELLA SP. (ICACINACEAE) 21
3.1 Introduction 21
3.2 Results and Discussion 21
IV 3.3 Experimental Section 30
3.3.1 General Experimental Procedure 30
3.3.2 Cytotoxicity Bioassays 31
3.3.3 Plant Material 31
3.3.4 Extraction and Isolation 31
IV. PENTOPETIA ANDROSAEMIFOLIA (APOCYNACEAE) 35
4.1 Introduction 35
4.2 Results and Discussion 35
4.3 Experimental Section 44
4.3.1 General Experimental Procedure 44
4.3.2 Cytotoxicity Bioassays 44
4.3.3 Plant Material 44
4.3.4 Extraction and Isolation 44
V. PHYSALINS FROM PHYSALIS ANGULATA (SOLANACEAE)) 48
5.1 Introduction 48
5.2 Results and Discussion 48
5.3 Experimental Section 59
5.3.1 General Experimental Procedure 59
5.3.2 Cytotoxicity Bioassays 59
5.3.3 Plant Material 59
5.3.4 Extraction and Isolation 59
VI. ROUPELLINA BOIVINII (APOCYNACEAE) 65
6.1 Introduction 65
V 6.2 Results and Discussion 69
6.3 Experimental Section 78
6.3.1 General Experimental Procedure 78
6.3.2 Cytotoxicity Bioassays 78
6.3.3 Plant Material 78
6.3.4 Extraction and Isolation 79
VII. CUCURBITACINS FROM OCTOLEPIS AFF. DIOICA (THYMELAEACEAE) 82
7.1 Introduction 82
7.2 Results and Discussion 82
7.3 Experimental Section 88
7.3.1 General Experimental Procedure 88
7.3.2 Cytotoxicity Bioassays 88
7.3.3 Plant Material 88
7.3.4 Extraction and Isolation 88
VIII. OTHER PLANTS STUDIED, BUT DROPPED 93
PART II
I. A SYNTHETIC APPROACH TO LUCILACTAENE 95
1. Introduction 95
1.1 The Cell Cycle as a Target for Anticancer Drugs 96
1.2. Cell Cycle Inhibitors 96
1.3 The p53 Tumor Suppressor Gene 97
1.4 Lucilactaene, a New Cell Cycle Inhibitor 98
VI 2. Results and Discussion 101
2.1 Retrosynthesis 101
2.2 Synthetic Aproach 102
2.2.1 Fragment II 102
2.2.2. Fragment IV 105
2.2.3 Fragment V 107
2.2.4 Fragment III 108
2.2.5. Coupling fragment III and V 112
2.2.6. Suggested work: Enzymatic lactonization 114
3. Conclusion 115
4. Experimental Section 115
VII List of Figures
Figure 1.1 Sample loading in a typical microtiter plate cytotoxicity assay 10
Figure 2.1 Biossay guided fractionation of Craspidospermum verticillatum 20
Figure 3.1 Structure and HMBC correlations of compound 3.1 22
Figure 3.2 Structure and HMBC correlations of compound 3.2 24
Figure 3.3 Bioassay guided fractionation of Casimirella sp. 32
Figure 4.1 HMBC correlation compound 4.3 40
Figure 4.2 Bioassay guided fractionation of Pentopetia androsaemifolia Decne 41
Figure 5.1 Bioassay guided fractionation of Physalis angulata L 64
Figure 6.1 Structure, ROESY and HMBC correlations of compound 6.1 67
Figure 6.2 Structure, ROESY and HMBC correlations of compound 6.2 69
Figure 6.3 Structure, ROESY and HMBC correlations of compound 6.3 71
Figure 6.4 Bioassay guided fractionation of Roupellina boivinii 80
Figure 7.1 Bioassay guided fractionation of Octolepis aff. Dioica Capuron (MG 985) 91
Figure 7.2 Bioassay guided fractionation of Octolepis aff. Dioica Capuron (MG 988) 92
Figure II.1 Cell cycle 96
VIII List of Schemes
Scheme 1 Retrosynthesis of lucilactaene 101
Scheme 2 Synthesis of compound 5 102
Scheme 3 Synthesis of α,β-unsaturated ester 6 103
Scheme 4 Synthesis of fragment II 104
Scheme 5 Synthesis of fragment IV 106
Scheme 6 Synthesis of fragment V 108
Scheme 7.1 Coupling Fragment V and IV 109
Scheme 7.2 Removal of the silyl group 111
Scheme 8 Synthesis of tetraenes 30a and 30b 112
Scheme 9 Trityl deprotection 113
IX List of Tables
13 Table 2.1 C NMR Data of 2.1, 2.3, and 2.4 in CDCl3 15
1 13 Table 3.1 H and C NMR data in C5D5N for Compounds 3.1-3 29
13 Table 3.2 C NMR data in DMSO-d6 for compound 3.4 -3.8 30
Table 4.1 1N NMR data for the sugar portion of compound 4.3 40
Table 4.2 13C NMR data of compounds 4.1, 4.2, 4.4 42
Table 4.3 13C NMR data of compounds 4.3 43
Table 5.1 13C NMR data of compounds 5.1-5.4 57
Table 5.2 13C NMR data of compounds 5.5-5.7 58
Table 6.1 13C NMR data of compounds 6.1-6.3 76
Table 6.2 13C NMR data of compounds 6.4-6.6 77
Table 7.1 13C NMR data of compounds 7.1-7.3 87
X PART I
I. INTRODUCTION 1.1 Cancer
Since 1990, more than 17 million new cancer cases have been detected in the US alone, not including carcinoma, basal cell and skin cancers.1 With all the efforts to fight this deadly
disease, the number of people diagnosed has not decreased. The National Cancer Institute (NCI)
predicts that 1,372,910 new cancer cases will be diagnosed in the US in 2005 and that 570,280
Americans are expected to die of the disease.1 The following estimated numbers in selected
cancers are expected in 2005: breast cancer (211,240 new cases and 40,870 expected deaths),
lung and bronchial cancer (172,570 new cases and 163,510 deaths expected); these figures
represent about 13% of all cancer diagnoses and account for 28% of all cancer deaths, and
prostate cancer (232,090 new cases and 30,350 deaths expected).1 The National Institutes of
Health (NIH) estimated that the overall costs for cancer in 2004 were $189.8 billion: $60.4 billion for direct medical costs, $16.9 billion for indirect morbidity costs and $95.2 billion for indirect mortality costs.1 These numbers are a clear indication that cancer affects all aspects of society. The statistics above are for the U.S.A alone. The main goal of the American Cancer
Society is to eradicate the disease, and the society predicts a reduction in the number of cancer cases by one half by the year 2015.
1.2 Importance of natural products to drug discovery
For many decades, synthetic chemicals as drugs have been effective in the treatment of most diseases. The pharmaceutical industry has synthesized over 3 million new chemicals in
1 Cancer Fact and Figures 2005 www.americancancersociety.org ; Visited on June 14, 2005
1 their effort to produce new drugs. Despite their success in developing drugs to treat or cure many
diseases, the treatment of certain diseases such as cancer, AIDS, heart disease and diabetes has
not been a complete success due to the complexity of these diseases.
Over the centuries, people have been living in close association with the environment and
relying on its flora and fauna as a source of food and medicine. As a result, many societies have
their own rich plant pharmacopeias. In developing countries, due to economic factors, nearly
80% of the population still depends on the use of plant extracts as a source of medicine.
Natural products also play an important role in the health care system in developed
countries. The isolation of the analgesic morphine from the opium poppy, Papaver somniferum,
in 1816 led to the development of many highly effective pain relievers.2 The discovery of
penicillin from the filamentous fungus Penicillium notatum by Fleming in 1929 had a great
impact on the investigation of nature as a source of new bioactive agents.3 Natural products can also be used as starting materials for semisynthetic drugs. The main examples are plant steroids, which led to the manufacture of oral contraceptives and other steroidal hormones. Today, almost every pharmacological class of drugs contains a natural product or natural product analog.
The investigation of higher plants has led the discovery of many new drugs. So far only a small portion of higher plants has been investigated. Consequently, they still remain a big reservoir of useful chemical compounds not only as drugs, but also as templates for synthetic analogues.
1.3 Natural products as anticancer agents
The effort to find anticancer agents from higher plants was launched by the US National
Cancer Institute (NCI) in 1957. So far plants have been a proven source of useful antitumour
2 Benyhe, S.; Morphine: New aspects in the study of an ancient compound. Life Sci. 1994, 55, 969-979. 3 Bennett, J. W.; Chung, K. T. Alexander Fleming and the discovery of penicillin. Adv. Applied Micro, 2001, 49, 163-184
2 substances. Today, many of the most useful and curative anticancer drugs are derived from natural product sources. Since the initiation of the program by NCI, more than 35,000 plant species have been investigated. The investigation of plants by various groups has produced the discovery of anticancer drugs such as vincristine, vinblastine, taxol, indicine-N-oxide, etoposide and its analogs, camptothecin and its analogs, etc.
Camptothecin (1), isolated from Camptotheca acuminata, is too insoluble for drug use, but its analogues topotecan and irinotecan are used to treat gastric, rectal, colon, and bladder cancers.4,5 They act on both DNA and RNA synthesis by inhibiting the enzyme topoisomerase I, which results in protein-linked breakdown of DNA.6,7,8
R2 R2 R1 O N N O
O Irinotecan 2b R1 = O C N N R2 = H, R3 = CH2CH3
OH O
Camptothecin 1 R1 = R2 = R3 = H
Topotecan 2a R1 = OH R2 = CH2NMe2.HCl
Taxol (paclitaxel) (3) is used in the treatment of ovarian and breast cancers.9,10 It binds to the β-tubulin subunit of microtubules and stabilizes the microtubule to normal disassembly.11
This results in mitotic block and ultimately in cell death by apoptosis.12
4 Wall, M.E.; Wani, M.C.; Cook, C.E.; Palmer, K.H.; Mc Phail, A.T.; Sim, G.A. Plant antitumor agents: The isolation and structure of camptothecin, a novel alkaloids; leukemia and tumor inhibitor from Camptotheca acuminata. J. Am. Chem. Soc. 1966, 88, 3888-3890. 5 Arun, B; Frenkel, E P. Topoisomerase I inhibition with topotecan: Pharmacologic and clinical issues. Expert opin. Pharmacother. 2001, 2, 491-505 6 Gelderblom, H. A. J.; De Jonge, M. J. A.; Sparreboom, A.; Verweij, J. Oral topoisomerase I inhibitors in adult patients: present and future. Invest. New Drugs, 1999, 17, 401-415 7 Kantarjian, H. New development in the treatment of acute myeloid leukemia: focus on topotecan. Semin.hematol. 1999, 36, 16-25 8 Kollmannsberger, C.; Mross, K.; Jakob, A.; Kanz, L.; Bokemeyer, C. Topotecan, a novel topoisomerase I inhibitor. Pharmacology and clinical experience. Oncology , 1999, 56, 1-12 9 Wani, M.C.; Taylor, H.L.; Wall, M.E.; Coggon, P; Mc Phail, A.T. Plant Antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J. Am. Chem. Soc. 1971, 93, 2325-2327.
3 AcO O OH O
Ph NH O
Ph O H O OH OH OAc OBz Taxol 3
Vinblastine (4) and vincristine (5), isolated from Catharanthus roseus, are used to treat leukemia, bladder and testicular cancers. Their mode of action is to bind to tubulin and stop its polymerization into microtubules, thus blocking the cell division.13,14
OH N
N Vinblastine 4 R = CH3 N H H Vincristine 5 R = CHO H3COOC H3COC N OCOCH3 R COOCH3 Etoposide (6) and its thiophene analog, teniposide (7) are semisynthetic derivatives of the natural product epipodophyllotoxin (8), and are used clinically to treat small-cell lung cancer, testicular cancer, lymphomas and other cancers. 1516 They inhibit the enzyme DNA topoisomerase II and cause DNA cleavage.
10 Stein, C. A. Mechanism of action of taxanes in prostate cancer. Semin. Oncol. 1999, 26, 3-7 11 Yeung, Tai K.; Germond, C.; Chen, X.; Wang, Z. The mode of action Taxol: Apoptosis at low concentration and Necrosis at high concentration. Biochem .biophys .res. commun.1999, 263, 398-404 12 Fan, W. Possible mechanisms of paclitaxel-induced apoptosis. Biochem. Pharmacol. 1999, 57, 1215-1221 13 Ngan, Vivian K.; Bellman, Krista; Hill, Bridget T.; Wilson, Leslie; Jordan, Mary Ann. Mechanism of mitotic block and inhibition of cell proliferation by the semisynthetic vinca alkaloids vinorelbine and its newer derivative vinflunine. Mol. Pharmacol. 2001, 60, 225-232 14 Dhamodharan, R.; Jordan, M. A; Thrower, D.; Wilson, L. Vinblastine suppresses dynamics of individual microtubules in living interphase cells. Mol.Biol.Cell , 1995, 6,1215-1229 15 Van Manen, J.M.S.; Retel, I.; De Vries, J.; Pinedo, H.M. Mechanism of action of the antitumor drug etoposide: a review. J. Natl. Cancer Inst. 1988, 80, 1526-1533. 16 Huff, Anne C.; Kreuzer, Kenneth N. Evidence for a common mechanism of action for antitumor and antibacterial agents that inhibit type II DNA topoisomerases. J. Biol. Chem. 1990, 265, 20496-505
4 H
R O O O OH O O O O O O O O O
H3CO OCH3 H3CO OCH3 OCH3 OH Etoposide 6, R = CH 3 Epipodophyllotoxin 8 Teniposide 7, R = S
In view of this history of successful anticancer drug development from natural products, it is reasonable to believe that more antitumour compounds still exist in nature, and that they can be discovered with appropriate strategies, resources and effort.
1.4 Biodiversity loss and the ICBG Program
The first step of a natural product drug discovery study is selecting the plant species to be investigated. Most plant extracts come from tropical rain forests in developing countries. Today, due to economic factors, the rain forests with their valuable ecosystems are disappearing rapidly.
With the high price of modern medicine, many people in tropical countries still depend on the use of plant extracts as a source of medicine. The traditional healers in the third world acquire their knowledge of bioactive plants by living in intimate association with their environment.
These traditional medicinal plants should be investigated before the knowledge of these healers disappears. Western civilization is affecting the third world with road building, commercial activities, and missionary efforts. With western education, young people are leaving their villages for the cities, leaving behind a wealth of ethnopharmacological information. This knowledge is fading slowly if no action is taken to document it, and its treasure will be lost
5 forever. The traditional healers know exactly which kind and which parts of plants need to be
collected. Plants species are disappearing due to human activities. In many tropical countries the
core of their economy depends heavily on their tropical forests. Activities such as farming and
timber industries are having an impact on their rain forests. Drug discovery from botanical
sources demands the screening of many extracts, and the loss of plants and marine organisms is
thus a significant barrier to drug discovery.
The International Cooperative Biodiversity Group (ICBG) program was started in 1992
to promote drug discovery and biodiversity conservation via economic development in
underdeveloped countries. The objective of this program is to discover new drugs and to share
any benefit with host countries if a drug is discovered from their botanical sources. The ICBG
program at Virginia Polytechnic Institute and State University has focused on the two countries
of Suriname and Madagascar.
1.5. Bioassays in the discovery of antitumor agents
1.5.1 Role of assays
Bioassays are required to select crude materials and isolate potential new antitumor
agents from natural source. The assay must be reliable, reproducible, sensitive and predictive.17
To determine the true efficacy of potential anticancer agents, it is important to evaluate their potency in more advanced testing systems followed by preclinical trials. In vitro bioassays in the cancer area can be divided into two groups: molecular assays and cellular assays. Molecular assays look for activity using isolated systems such as enzymes, receptors, DNA, etc., while
17 Dey, P.M.; Harborne, J.B. Methods in plant biochemistry. Harborne, J.B (Ed); Academic Press Inc: San Diego, 1991; Volume 6: Assays for bioactivity. Pp 71-133
6 cellular assays use intact cells. Molecular assays usually deal with a single subcellular target,
while cellular assays detect any substance inhibiting cell growth.
The simultaneous advantage and disadvantage of molecular assays are their high
specificity. Any compound which acts by a different mechanism from the assay will not be
detected. The philosophy of molecular assays is not about what you miss but what you find, so
the “hit rate” is very low.
Cellular assays, sometimes referred to as cytotoxicity assays, deal with the inhibition of
cell growth and the death of cells. The problem with these assays is that active materials may
include heavy metals, detergents, sodium or calcium channel poisons, and non-specific oxidants.
All these elements can cause cell death. 18 It is therefore difficult to distinguish between promising antitumor agents and merely toxic agents.
1.5.2 Antitumor assays
There are many bioassays available for the isolation and identification of potential antitumor substances. The most commonly used assays are cytotoxic, antimitotic, and antimetastatic assays, and also assays based on interactions with DNA.
Cytotoxicity assays.17,19 In spite of the problems noted above, most of the research programs involving the isolation and identification of new cancer agents use cytotoxicity for bioassay-directed fractionation. The procedure involves treating cancer cells with different concentrations of the test material, then assessing cell growth after 48h or 72h of incubation. The most common cell types used are human cell lines derived from various human tumor types.
18 Olivieri, G.; Brack, Ch.; Muller-Spashn, F.; Mercury induces cell cytotoxicity and oxidative stress and increases b-amyloid secretion and tau phosphorylation in SHSY5Y neuroblastoma cells. J. Neurochem. 2000, 74, 231-236 19 Stammati, A.; Bonsi, P.; Zucco, F.; Moezelaar, R.; Toxicity of Selected plant volatiles in microbial and mammalian short-term assays. Food Chem. Toxicol. 1999, 37, 813-823
7 Antimitotic assays. 17,20 The human cytoskeleton is composed of fibrous elements such as actin and myosin, filaments and microtubules. These elements are involved in cell dynamics.
Microtubules are crucial to the division of cells. A change in microtubule function by chemical means stops cell division, and therefore prevents the proliferation of cells. Antimitotic agents such as vincristine and paclitaxel interact with microtubules. Vincristine inhibits the assembly process, while paclitaxel promotes the assembly of microtubules and inhibits depolymerization.
Tubulin can be extracted from brain (bovine, porcine etc.) and purified by repeated cycles of polymerization and depolymerization coupled with centrifugation. The microtubules are formed at 38 ºC in the presence of GTP and Mg2+ and depolymerize at 0 ºC. Observing light scattering at
350 nm can monitor the assembly reaction. The test substance can be added to an in vitro solution of tubulin; if it prevents the heat and GTP-induced polymeration of microtubules, the substance may be an antimitotic agent.
Antimetastatic assays.17,21,22 Metastasis is the process where malignant cells spread from the site of the primary tumor, via the circulatory system, to other sites throughout the body. A bioassay for antimetastatic activity has been developed which depends on the fact that tumor cells have the ability to invade and penetrate fresh amnion membranes obtained from human placenta. In the procedure, cancer cells are placed in the upper compartment of a closed chamber onto the amnion epithelial layer, either in the presence or absence of the test sample. An equivalent amount of the test substance must be added to the lower compartment of the chamber to prevent a change in concentration. Cell migration is monitored using a microscope. If the test
20 Roberge, M.; Cinel, B.; Anderson, H. J.; Cell-based screen for antimitotic agents and identification of analogues of rhizoxin, eleuthebin, and paclitaxel in natural extracts. Cancer Res. 2000, 60, 5052-5058 21 Kang, Y-H; Kim, W. H; Park, M. K; Han, B. H. Antimetastatic and antitumor effects of benzoquinonoid AC7-1 from Ardisia crispa. Int. J. Cancer, 2001, 93, 736-740 22 Reich, R.; Adler, S. H.; Martin, G. R.; Royce, L S. Use of in vitro assays to define the malignant activities of tumor cells and to screen for antimetastatic drugs. Altern. Methods Toxicol. 1989, 7,11-22
8 substance prevents the migration of tumor cells from one compartment to the other, the substance may be antimetastatic agent.17
Assays based on interactions with DNA.17,23,24 Many known antitumor agents act by interacting with DNA and changing its function or expression. A number of enzymes are required for the biological function of DNA. The enzymes topoisomerase I and II are able to cut either one strand of DNA (topoisomerase I) or a pair of complementary strands (topoisomerase II) and then reconnect the DNA backbone. These enzymes are important because DNA has to uncoil
(undergo a topological change) prior to replication.
1.6 A2780 Cytotoxicity Assay
The A2780 human ovarian cancer line used in this research was developed at the
National Cancer Institute (NCI).25 The procedure involves treating cancer cells with different concentrations of the test material, and then assessing cell growth after 48 hours under 5% CO2 at 37 ºC (Scheme 1). After 48 hours, the old media is removed and replaced with media containing 1 % Alamar Blue solution and the plate is allowed to stand for 4 hours.
Alamar Blue can adopt two forms: an oxidized form, blue in color, called resazurin, and a reduced form, which is a reddish pink color and which fluoresces, called resorufin (Figure 1.1).
The mechanism by which Alamar Blue is reduced by living cells and where the reduction takes place are still topics of debate. Some authors hold that Alamar Blue is reduced by mitochondrial activity and there are studies that support this theory. A recent study also indicated that the
23 Roa, H.; Stillman, B. The origin recognition complex interacts with a bipartite DNA binding site within yeast replicators. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 2224-8 24 Kerklaan, Peter R. M.; Bouter, Susan; Van Elburg, Paul E.; Mohn, Georges R. Evaluation of the DNA repair host-mediated assay. II. Presence of genotoxix factors in various organs of mice treated with chemotherapeutic agents. Mutat.Res. 1986, 164, 19-29 25 Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J.T.; Bokesch, H.; Kenny, S.; Boyd, M. R. New colorimetric cytotoxicity assay for anticancer-drug screening J. Natl. Cancer. Inst. 1990, 82, 1107
9 reduction may take place in the cell’s cytoplasm.26 In any event, the reduction of Alamar Blue by living cells to produce a fluorescent product provides a convenient way to determine cell viability after treatment with a cytotoxic agent.
The reduction of Alamar Blue can be monitored either by measuring absorbance or by measuring fluorescence. The fluorescence measurement is obtained by exciting the reduced form of Alamar Blue at 530-560 nm and determining emission at 590 nm; this is the method used in our cytotoxicity bioassays. After treatment with Alamar Blue, the plate is placed in a multi-well plate reader and the fluorescence of the cells is determined. The % of the cell growth inhibition is calculated based the fluorescence of the cells, using a simple algorithm. The sample wells are compared with controls wells (cells plus media). The IC50 value is then obtained by plotting the
% of cell growth inhibition against concentration.
TEST SAMPLE AT DIFFERENT CONCENTRATIONS POSITIVE CONTROL: PLUS CELLS CELLS, MEDIA, ACTINOMYCIN D
O 20 + N 5 + O O OH 1.25 + Resazurin 0.3125 + _ 0.07813 _ RPMI MEDIUM 0.01953 + 10% FBS N _ 0.00488 O O OH 0.00122 _ Resorufin
NEGATIVE CONTROL: CELLS, MEDIA Figure 1.1: Sample loading in a typical microtiter plate cytotoxicity assay
26 O’Bbrien, J.; Ian, W.; Terry, O.; François, P. Investigation of the Alamar Blue (reazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J. Biochem. 2000, 267, 5421-5426
10 II. INDOLE ALKALOIDS FROM CRASPIDOSPERMUM VERTICILLATUM
2.1 Introduction
Craspidospermum verticillatum Bojer ex A. DC. belongs to the family Apocynaceae, which is known to contain alkaloids. The extract was collected in Madagascar via the ICBG program in order to investigate any potential anticancer activity. Its leaves and bark have been used by the local people to treat cough, stomach ache and syphilis.
2.2 Results and discussion
Ethanol extract from the roots and bark of Craspidospermum verticillatum Bojer ex. A.
DC. was partitioned between hexane and 60% aqueous MeOH, then partitioned between CH2Cl2
and 50% aqueous MeOH. All the fractions were tested for their cytotoxicity, and only the hexane
fraction was active. The active fraction was subjected to normal and reversed phase preparative
TLC and four known indole alkaloids, tabersonine (2.1),27,28 11-hydroxy-tabersonine (2.2),2,29
11-methoxy-tabersone (2.3),1 and vandrikine (2.4)30 were isolated.
Compound 2.1 was isolated as an amorphous substance. Its 1H NMR spectrum revealed the presence of a triplet at δ 0.64 (J = 7.5 Hz, 3H) and a doublet of quartets at δ 0.85 and 0.99 (J
= 15.0, 7.5, 2H) which indicated the presence of a CH3CH2- group. A singlet peak at δ 3.77 (3H)
showed the presence of one methoxy group. A doublet of triplets and doublet of doublets at δ
5.71 (J = 10.0, 1.3, 1H) and 5.79 (J = 10.0, 3.6, 1.3, 1H) respectively also indicated the presence
of an internal double bond.
27 Guo, L; Zhou, Y. Alkaloids from Melodinus hemsleyanus. Phytochemistry, 1993, 34, 563-6 28Ye, J; Zhou, Y.; Huang, Z; Picot, Francoise. Alkaloids from Melodinus suaveolens. Phytochemistry, 1991, 30, 3168-70. 29 Kan-Fan, C.; Das, B. C.; Potier, P.; Le Men, J.; Boiteau, P. Madagascan plants. I. Craspidospermum verticillatum alkaloids. Ann. Pharm. Fr., 1968, 26, 577-82 30 Verpoorte, R.; Van Beek, T. A.; Riegman, R. L. M.; Hylands, P. J.; Bisset, N. G. Aromatic chemical shifts in ar- hydroxy- and -methoxy-substituted indole alkaloids; reference data and substituent-induced chemical shifts for ten different chromophoric groups. Org. Magn. Reson., 1984, 22, 328-35.
11 The 1H NMR spectrum exhibited peaks for four aromatic protons at δ 6.82 (d, J = 7.5 Hz),
6.87 (t, J = 7.5), 7.14 (t, J = 7.5 Hz) and 7.24 (d, J = 7.5 Hz). The spectrum also showed a peak at δ 8.99 (s). The 13C NMR spectrum exhibited peaks for 21 carbons, which included a carbonyl peak at δ 169.0, and also confirmed the presence of an aromatic residue. Its molecular formula was determined by HRFABMS as C21H25N2O2. The NMR data of compound 2.1 combined with its elemental composition indicated that it was most probably an indole alkaloid of the tabersonine class. A comparison of its NMR data with the literature data for tabersonine31 (Table
2-1) indicated that compound 2.1 is tabersonine. Compound 2.1 had an IC50 of 1.5 µg/mL in the
A2780 ovarian cancer cell line.
3 5 N 14 9 6 21 20 10 8 15 7 H 18 11 17 19 N 2 16 12 H OOCH3
2.1
Compound 2.2 was isolated as an amorphous substance. Its 1H NMR spectrum was
identical to the spectrum of compound 2.1 except that it exhibited only three aromatic protons at
δ 7.04 (d, J = 7.9 Hz, H-9), 6.32 (d, J = 7.9 Hz, H-10) and 6.35 (s, H-12). The observation of a
singlet aromatic proton indicated that the substituent on the aromatic ring must be at C-10 or C-
11. The molecular formula was determined to be C21H24N2O3 by HRFABMS. This compound
has one additional oxygen atom as compared with compound 2.1. The only major differences in
the NMR spectra of compounds 2.1 and 2.2 were observed in the aromatic ring signals,
suggesting that compound 2.2 is 10- or 11- hydroxytabersonine. There are no reports of an indole
31 Wenkert, E.; Cochran, D. W; Hagaman, E. W.; Schell, F. M. Neuss, N.; Katner, A. S.; Potier, P.; Kan, C.; Plat, M.; Koch, M.; Mehri, H.; Poisson, J.; Kumesch, N.; Rolland, Y.; Carbon -13 nuclear magnetic resonance spectroscopy of naturally occurring substances. XIX. Aspidosperma alkaloids. J. Am. Chem. Soc. 1973, 15, 4990- 4995
12 alkaloid of the tabersonine class with a hydroxyl group at the C-10 position, so that possibility
was unlikely. A firm distinction between these two isomers was made by comparison of the 1H
NMR spectrum of compound 2.2 with the 1H NMR of 11-hydroxyvincadifformine.32 Based on
these comparisons the structure of compound 2.2 was determined to be 11-hydroxytabersonine,
which was first isolated in 1968. The 13C NMR data could not be obtained because the
compound decomposed in CDCl3 sitting in the NMR tube overnight. Although the compound
has been mentioned in several publications, its 1H and 13C NMR data have not been published. It
had an IC50 of 2.8 µg/mL against the A2780 ovarian cancer cell line.
N
H HO N H OOCH3 2.2
Compound 2.3 was also isolated as an amorphous substance. Its 1H NMR spectrum was
identical to the spectrum of compound 2.2 except that it had a signal for an additional methoxy
group at δ 3.7. This group was located at C-11 by similar reasoning to that for compound 2.2. Its
molecular formula was determined by HRFABMS as C22H27N2O3. Its structure was confirmed by using 1H NMR and 13C NMR spectra, and also by comparing its 13C NMR data and MS to the
31 published data of 11-methoxytabersonine (Table 2-1). It had an IC50 of 0.9 µg/mL against the
A2780 cell line.
32 Guo, L. W.; Zhou, Y. L. Alkaloids from Melodinus hemsleyanus, Phytochemistry , 1993, 34, 563-566
13 N
H
H3CO N H OOCH3
2.3
Compound 2.4 was also isolated as an amorphous substance. The 1H NMR spectrum was
identical to the spectrum of compound 2.3 except that the peaks at δ 5.71 and 5.79 for the C-
14/C-15 double bond were missing. In addition the signals for the C-18/C-19 ethyl group were
replaced by a doublet of doublets at δ 3.66 (dd, J = 4.1, 2.3 Hz, 2H) and a triplet at δ 0.93δ (t, J =
7.5 Hz, 2H). A new triplet at δ 3.65 (t, J = 7.5 Hz, 2H) was also observed. These observations suggested that the C-18/C-19 ethyl group had become oxygenated and cyclized onto C-15. The molecular formula was determined by HRFABMS as C22H27N2O4. The NMR spectra and
composition were consistent with those of the alkaloid vandrikine,31 and a comparison of 13C
NMR data with literature data showed that compound 2.4 is vandrikine. It had an IC50 of 3.8
µg/mL against the A2780 cell line.
N
O H
H3CO N H OOCH3 2. 4
14 13 Table 2.1 C NMR Data of 2.1, 2.3, and 2.4 in CDCl3
Carbon 2.1 Lit. values 31 2.3 Lit. values 31 2.4 Lit.values 31 2 166.8 166.7 167.2 167.4 167.8 167.4 3 50.6 50.3 50.7 50.8 46.0 45.7 5 50.9 50.8 51.0 51.2 51.5 51.1 6 44.3 44.3 44.6 44.8 45.3 45.1 7 55.1 55.0 54.5 54.8 54.5 54.2 8 138.1 137.8 130.5 130.8 130.3 130.5 9 121.5 121.4 121.8 122.0 121.7 121.5 10 120.6 120.5 105.0 105.3 105.1 104.8 11 127.7 127.6 160.0 160.3 160.1 159.8 12 109.3 109.2 96.9 96.7 96.8 96.5 13 143.1 143.1 144.4 144.7 144.4 144.0 14 124.9 124.8 124.9 125.1 27.6 27.4 15 133.1 133.1 133.1 133.4 80.0 79.8 16 92.2 92.2 92.4 92.7 94.2 93.9 17 26.9 26.7 26.9 27.2 26.9 26.6 18 7.5 7.3 7.5 7.7 65.0 64.7 19 28.5 28.4 28.4 28.8 34.9 34.6 20 41.3 41.2 41.4 41.7 46.7 46.4 21 70.0 69.9 70.2 70.4 68.9 68.7 C=O 169.0 168.8 169.0 169.2 168.8 168.5 OMe 51.1 50.8 51.0 51.2 51.1 50.8 OMe 55.5 55.7 55.5 55.2
2.3 Experimental Section
2.3.1 General Experimental Procedures
The NMR spectra were obtained on either a JEOL Eclipse 500 spectrometer or Varian
INOVA 400 spectrometer. The mass spectra were obtained on a JEOL JMS-HX-110 instrument.
The normal phase preparative TLC plate used was a 20 x 20 cm uniplate with silica gel GF layer with UV 254, 20 x 20 cm 500 μM thick obtained from Analtech. The reversed phase preparative
15 TLC plate used was Baker Si-C18 F TLC plate reversed phase octadecyl (C18), 20 x 20 cm 200
μM obtained from J.T.Baker.
2.3.2 Cytotoxicity Bioassays
The A2780 human ovarian cancer cell line cytotoxicity assay was performed at Virginia
Polytechnic Institute and State University by Jennifer Schilling as described below.
200 μL of RPMI was distributed to the column 12 well in a 96 well tissue culture plate.
20 μL RPMI media was added to column 11. 180 μL of 2.7 x 10-5 c/mL A2780 DDP-S (Platinol-
Sensitive cells (5 x 10-4 cells/well) were added to all wells in column 1 to 11. Plates were incubated for 3 hours in 5% CO2 at 37 ºC to allow cells to adhere.The diluted sample (20 μL) in aqueous DMSO (50 %) was added to the wells. Column 12 was used for positive media control.
Actinomycin D was the positive control media and was run at 4 dilutions with an IC50 ~1-3 ng/mL in column 11. RPMI (20 μL) was added to the last 4 rows of column 11 as a negative control. The plate was then incubated for 48 hours at 37 ºC in a 5 % CO2 incubator. After 48 hours, the old media was removed and replaced with 200 μL/well of fresh RPMI plus 10% Fetal
Bovin Serum (FBS) containing 1 % Alamar Blue solution. The plate was then incubated again for 4 hours at 37 ºC in 5 % CO2. The plates were placed in a multi-well plate reader and the fluorescence of the cells was determined. The fluorescence measurement was obtained by exciting the reduced form of Alamar Blue at 530-560 nm and determining emission at 590 nm.
The % of the cell growth inhibition is calculated based on the fluorescence of the cells, using a simple algorithm. The sample wells are compared with controls wells (cells plus media). The
IC50 value is then obtained by plotting the % of cell growth inhibition against concentration.
16 2.3.3 Plant Material
Root, bark, wood, twigs, leaves and inflorescence were collected by Chris Birkinshaw and his colleagues from the rainforest of Toamasina, Madagascar in the vicinity of the Zahamena
National Park on December 15, 1999.
2.3.4 Extraction and Isolation
The extract from the roots and bark of Craspidospermum verticillatum (1.27 g) was partitioned between hexane and 60% aqueous MeOH, then partitioned between CH2Cl2 and 50% aqueous MeOH. All the fractions were tested for their cytotoxicity, and only the hexane fraction was active. The hexane fraction (50.6 mg) was applied to a normal phase preparative TLC (20 x
20 cm) and developed using CHCl3:CH3CN (9:1) as mobile phase. Three bands were eluted and the eluants from the first two bands were reapplied to a reversed phase preparative TLC plate (20 x 20 cm and developed using CH3CN/H2O as mobile phase. These procedures yielded the pure compounds 2.1-2.4. Details of the fractionation are shown in Figure 2-1: Compound 2.1 was obtained as fraction 1-3-1, compound 2.2 as fraction 1-3-2, compound 2.3 as fraction 1-4 and compound 2.4 as fraction 2-2.
1 Compound 2.1: yellow amorphous substance (2 mg); H NMR (500 MHz, CDCl3) δ
0.64 (t, J = 7.5 Hz, 3H), 0.85 (dq, J = 15.0, 7.5 Hz, 1H), 0.99 (dq, J = 15.0, 7.5 Hz, 1H), 1.80 (dd,
J = 11.5, 4.5 Hz, 1H), 2.07 (td, J = 11.5, 6.5 Hz, 1H), 2.44 ( d, J = 15.0 Hz, 1H), 2.55 (dd, J =
15.0, 1.6, 1H), 2.68 (s, 1H), 2.72 (m, 1H), 3.05 (t, J = 7.4 Hz, 1H), 3.18 (d, J = 16.0 Hz, 1H),
3.47 (dd, J = 16, 4.5 Hz, 1H), 3.77 (s, 3H), 5.71 (dt, J = 10, 1.3 Hz, 1H), 5.79 (ddd, J = 10.0, 3.6,
1.3, 1 H), 6.82 (d, J = 7.5), 6.87 (t, J = 7.5 Hz, 1H), 7.14 (t, J = 7.5 Hz, 1H), 7.24 (d, J = 7.5 Hz,
17 13 1H), 8.99 (br s, 1H); C NMR (500 MHz, CDCl3, Table 2.1). HRFABMS found m/z 337.2018
+ [M+H] calcd for C21H25N2O2 337.2022.
1 Compound 2.2: yellow amorphous substance (1.3 mg); H NMR (500 MHz, CDCl3) δ
0.64 (t, J = 7.5 Hz, 3H), 0.85 (dq, J = 15.0, 7.5 Hz, 1H), 0.99 (dq, J 15.0, 7.5 Hz, 1H), 1.80 (dd, J
= 11.5, 4.5 Hz, 1H), 2.07 (td, J = 11.5, 6.5 Hz, 1H), 2.44 ( d, J = 15.0 Hz, 1H), 2.55 (dd, J = 15.0,
1.6, 1H), 2.68 (s, 1H), 2.72 (m, 1H), 3.05 (t, J = 7.4 Hz, 1H), 3.18 (d, J = 16.0 Hz, 1H), 3.47 (dd,
J = 16, 4.5 Hz, 1H), 3.77 (s, 3H), 5.71 (dt, J = 10, 1.3 Hz, 1H), 5.79 (ddd, J = 10.0, 3.6, 1.3, 1 H),
6.32 (d, J = 7.9 Hz, 1H), 6.35 (s, 1H), 7.03 (d, J = 7.5 Hz, 1H), 8.86 (br s, 1H); HRFABMS
+ found m/z 353.1896 [M+H] ; calcd for C21H25N2O3 353.1900.
1 Compound 2.3: yellow amorphous substance (6 mg); H NMR (500 MHz, CDCl3) δ 0.64 (t,
J = 7.5 Hz, 3H), 0.85 (dq, J = 15.0, 7.5 Hz, 1H), 0.99 (dq, J =15.0, 7.5 Hz, 1H), 1.80 (dd, J =
11.5, 4.5 Hz, 1H), 2.07 (td, J = 11.5, 6.5 Hz, 1H), 2.44 ( d, J = 15.0 Hz, 1H), 2.55 (dd, J = 15.0,
1.6, 1H), 2.68 (s, 1H), 2.72 (m, 1H), 3.05 (t, J = 7.4 Hz, 1H), 3.18 (d, J = 16.0 Hz, 1H), 3.47 (dd,
J = 16, 4.5 Hz, 1H), 3.77 (s, 3H), 5.71 (dt, J = 10, 1.3 Hz, 1H), 5.79 (ddd, J = 10.0, 3.6, 1.3, 1 H),
6.35 (d, J = 8 Hz, 1H), 6.42 (s, 1H), 7.11 (d, J = 7.5 Hz, 1H), 8.96 (br s, 1H). 13C NMR (500
+ MHz, CDCl3, Table 2.1). HRFABMS found m/z 367.2019 [M+H] ; calcd for C22H27N2O3,
367.2022.
1 Compound 2.4: yellow amorphous substance (6 mg); H NMR (500 MHz, CDCl3) δ 0.93 (t,
J = 7.5 Hz, 2H), 1.30 (m, 2H), 1.46 (m, 2H), 1.55 ( dd, J = 14.0, 7.5, 2H), 1.75 (dd, J = 11.5, 4.5
Hz, 1H), 2.03 (td, J = 11.5, 6.5 Hz, 1H), 2.26 ( d, J = 15.0 Hz, 1H), 2.30 (dd, J = 15.0, 1.6, 1H),
18 2.78 (s, 1H), 2.63 (m, 1H), 2.93 (t, J = 7.4 Hz, 1H), 2.96 (d, J = 16.0 Hz, 1H), 3.65 (t, J = 7.5 Hz,
1H), 3.67 (dd, J = 4.1, 2.3, 2H), 3.78 (s, 3H), 3.77 (s, 3H), 6.39 (d, J = 8 Hz, 1H), 6.4 (s, 1H),
13 7.11 (d, J = 7.5 Hz, 1H), 8.76 (br s, 1H). C NMR (500 MHz, CDCl3, Table 2.1).
+ HRFABMS found m/z 383.1968 [M+H] ; calcd for C22H27N2O4, 383.1971.
19 Crasspidospermum verticillatum (Apocynaceae)
MGO96
IC50 = 18.2 mg/mL 1.276 g
Partition:hexane/ 60%aq.MeOH
Partition:CH2Cl2/50%aq.MeOH Hexane 50.6 mg CH Cl MeOH % 4 2 2 1.04g IC50 (mg/mL) = 3.8 158.1 mg % 83.1 % 12.4 IC50 ( mg/mL) = 15.8 >20 Normal phase PTLC CHCl3/Acetonitrile 90/10
1 23 25.4 mg 12.2 mg 10.5 mg
IC50 (mg/mL) 3.0 Reverse phase PTLC 16.2 Acetonitrile/H2O 65/35
2-12-2 4.3 mg 5.2 mg Reverse phase PTLC Acetonitrile/H2O 65/35 35.2% 42.6
IC50 (mg/mL) 4.33.8
1-1 1-21-31-41-5
9 mg 2.4 mg 4.6 mg 6 mg 1.9 mg % 35.43 9.4 18.1 23.6 7.5
IC50 (mg/mL) > 20 15 3.2 0.9 11.9 TLC
1-3-1 1-3-2 2 1.3
IC50 (mg/mL) 1.5 2.8
Figure 2.1 Biossay guided fractionation of Craspidospermum verticillatum
20 III. CYTOTOXIC DITERPENOIDS FROM A CASIMIRELLA SPECIES
3.1 Introduction
The extracts from a Casimirella sp. were obtained from Suriname through the
International Cooperative Biodiversity Group (ICBG) in order to investigate for potential
cytotoxicity. The collection was sterile and could not be positively identified to species. This
investigation has afforded three new and five known cytotoxic diterpenoids.
3.2 Results and Discussion
The MeOH and EtOAc extracts of Casimirella sp. were partitioned between hexane and
60% aqueous MeOH, then partitioned between CH2Cl2 and 50% aqueous methanol. All the
fractions were tested for cytotoxicity, but only the CH2Cl2 fraction was active. The active
fraction was subjected to reversed phase C-18 column chromatography, followed by reversed
phase HPLC to produce the three new diterpenoids 3.1-3.3, as well as the five known diterpenoids 3.4-3.8.
Compound 3.1 was isolated as a white amorphous solid, and HRFABMS indicated a
1 molecular formula of C20H25O6. The H NMR spectrum (C5D5N) of compound 3.1 revealed the presence of two methyl groups as singlets at δ 1.0 and δ 1.56 corresponding to the methyl protons on C-17 and C-18, respectively. The spectrum also showed multiplets at δ 1.62, δ 2.22 and 2.25, δ 1.18 and 1.58, and δ 1.32 for C-1, C-2, C-11, and C-12 methylenes respectively, and also two doublets of doublets at δ1.68 (dd, J = 12.1, 2.9 Hz), δ 2.34 (dd, J = 2.6, 6.9 Hz) for C-9, and C-5 methine respectively. The spectrum had a pair of doublets at δ 3.68 (d, J = 3.4, 5.9 Hz) and 4.31 (d, J =3.4, 5.9 Hz), and also at δ 3.97(d, J = 17.2 Hz) and δ 4.53 (d, J = 17.2 Hz) assigned to the methylene protons on C-20 and C-16, respectively. The spectrum also exhibited a
21 singlet at δ 4.15, a doublet of doublets at δ 4.98 (dd, J = 2.3, 4.8 Hz), another doublet at δ 6.10 (d,
J = 4.9 Hz) and another singlet at δ 7.02 corresponding to the C-14 and C-6 methines, the C-7 olefinic proton and the hydroxylic proton on C-3, respectively. The 13C NMR spectrum of compound 3.1 showed the presence of 20 carbons (Table 3-1). It had one carbonyl signal at δ
216.18 assigned as C-15 and one lactone signal at δ 179.24 assigned as C-19. The carbon signals
at δ 119.22 and δ 144.32 corresponding to C-7 and C-8, respectively, indicated the presence of
one double bond. The DEPT spectrum revealed that its carbon skeleton was composed of two
methyls, six methylenes, five methines and seven quaternary carbons. The final structure was
elucidated using COSY, HMQC, HMBC, and NOESY correlation spectra (See Appendix). The
structure was confirmed by comparing its 2-D data to the published 2-D data of the known
compound 3.4. 33 Both compounds have the same connectivity and correlations. The only
difference between the new compound 3.1 and the known one is that the C-17 (Me) showed a
HMBC correlation to a ketone (C-15) at δ 216.18 while C-17 for the known compound 3.4
showed a correlation to the C-15 carbinol carbon. That difference confirmed the position of the
carbonyl at the C-15 position.
17 O O OH 11 15 20 13 1 16 9 O O O O O H H O H H H H 3 5 6 7 HO HO HO H H H H H H 18 O O O O O 19 O 3.4 3.1
Figure 3.1 Structure and HMBC correlations of compound 3.1
33 Graebner, I. B.; Mostardeiro, M. A.; Ethur, E. M.; Burrow, R. A.; Dessoy, E. C. S.; Morel, A. F. Diterpenoids from Humirianthera ampla. Phytochemistry, 2000, 53, 955-959.
22 Compound 3.2 was isolated as a colorless amorphous solid, and HRFABMS indicated the
1 molecular formula of C20H25O7. The H-NMR spectrum of compound 3.2 also revealed the
presence of two methyl singlets at δ 1.03 and 1.60, corresponding to the C-17 and C-18 methyl
protons, respectively. The spectrum also exhibited multiplets at δ 1.18 and 1.58 for C-11 and at δ
1.32 for the C-12 methylene protons, as well as two doublets of doublets at δ 2.41 (dd, J =2.6, 7
Hz) and δ 2.70 (dd, J = 2.6, 7 Hz) for C-9 and C-5 methines, respectively. The proton spectrum
showed a doublet of doublets at δ 2.99 (dd, J = 4, 4) for the C-2 methylene. It also revealed the
presence of doublets at δ 3.96 (d, J = 16.9) and δ 4.47 (d, J = 16.9 Hz) assigned to the C-16 methylene protons and a doublet of doublets at δ 4.33 and 4.64 (dd, J = 3.4, 8.9 Hz) corresponding to the C-20 methylene protons. The spectrum also had a singlet at δ 4.19 and a doublet of doublets at δ 4.24 (dd, J = 1.9 3.9 Hz) assigned to the C-14 and C-1 methine protons, respectively, as well as a doublet at δ 6.16 (d, J = 4.6 Hz) and a doublet of doublets at δ 5.13 (dd,
J = 2.3, 4.8 Hz) corresponding to the C-7 and C-6 methine protons, respectively. The spectrum exhibited a singlet at δ 6.90 and a doublet at δ 6.87 (d, J = 4.0 Hz) for hydroxylic protons on C-3 and C-1. Its 13C-NMR spectrum also indicated the presence of 20 carbons including a carbonyl ketone at δ 216.7 assigned to C-15, and a carbonyl lactone at δ 179.38 assigned to C-19 (table 3-
1). Its DEPT spectrum revealed that the skeleton was made of two methyls, five methylenes, six methines, and seven quaternary carbons. The final structure was elucidated using COSY, HMQC,
HMBC and NOESY correlation spectra. Compound 3.2 has the same connectivity and correlations as compounds 3.1 and 3.4. The position of the 1-β-OH was determined based on the
HMBC correlation depicted in Figure 3.2.
23 O O OH OH O O O H H O H H HO HO H H H H O O O O 3.2
Figure 3.2 Structure and HMBC correlations of compound 3.2
Compound 3.3 was isolated as a colorless amorphous solid, and HRFABMS indicated
1 molecular formula of C20H27O6. The H NMR spectrum of compound 3.3 also revealed the presence of two methyl singlets at δ 1.04 and 1.55 corresponding to the C-17 and C-18 methyl protons, respectively. It also showed multiplets at δ 1.64 and 1.71, δ 2.22 and 2.25, δ 1.18 and
1.61 and δ 1.21 for C-1, C-2, C-11 and C-12 methylenes, respectively. It also exhibited two doublet of doublets at δ 2.41 (dd, J = 2.6, 7.0 Hz) and δ 2.70 (dd, J = 2.6, 7.0 Hz) for C-9 and C-
5 methines respectively. The proton spectrum showed doublet of doublets at δ 4.33 (dd, J =3.4,
5.9) and δ 4.64 (dd, J = 3.4, 8.9 Hz), a doublet of doublets at δ 3.96 (dd, J = 16.9 Hz) and δ 4.47
(dd, J = 16.9 Hz) assigned to the C-20 and C-16 methylene protons, respectively, and a singlet at
δ 3.91 for C-14 methine proton. The spectrum exhibited a doublet at δ 5.95 (d, J = 4.8 Hz) for the
C-7 methylene protons, a doublet of doublets at δ 4.96 (dd, J = 4.8, 6.8 Hz) for the C-6 methylene protons, and a doublet of triplets at δ 4.05 (t, J = 8.1 Hz) and 4.23 (t, J = 8.1 Hz). The spectrum also exhibited a doublet at δ 6.82 and a singlet at δ 6.83 for the hydroxylic protons on
13 C-15 and C-3 respectively. The C NMR (C5D5N) spectrum indicated the presence of 20 carbons including a lactone carbon signal at δ 179.53 assigned to C-19 (Table 3.1). Its DEPT spectrum revealed that the skeleton consisted of two methyls, six methylenes, six methines, and
24 six quaternary carbons. The final structure was elucidated using COSY, HMQC, HMBC and
NOESY correlation spectra. The same 2-D correlations were observed as for compound 3.4.
OH
O O H H HO H H O O 3.3
Compounds 3.3 and 3.4 both had a molecular formula of C20H26O6, and both displayed
the same number of methylene, methine and quaternary carbons, but there is a clear difference in
their carbon spectra, particularly at the chemical shifts of C-13, C-14, C-15, C-16 and C-17. The
chemical shift for C-13 is at δ 49.99 for compound 3.4 and δ 47.25 ppm for compound 3.3, C-14
at δ 86.46 for compound 3.4 and at δ 88.08 for compound 3.3, C-15 at δ 78.76 for compound 3.4
and δ 79.36 for compound 3.3, C-16 at δ 76.38 for compound 3.4 and δ 72.68 for compound 3.3,
and C-17 at δ 15.64 for compound 3.4 and δ 19.26 for compound 3.3. The absolute configuration
of C15 of the known compound 3.4 was previously assigned as 15 (S) by the Horeau method.33
This assignment, taken in conjunction with an X-ray structure determination of the 15-acetate,
which gave the relative stereochemistry of all the stereocenters determined the abosolute
stereochemistry of the compound to be 3(S), 4(R), 5(R), 6(R), 10(R), 13(R), 14(R) and 15(S).34
As already mentioned, compound 3.4 was a known substance and its 1H and 13C NMR spectra agreed with the reported data for humirianthol in DMSO-d6. Compound 3.4 was identified as humirianthol (Table 3.1).
34 Burrow, R. A.; Morel, A. F.; Graebner, I. B.; Farrar, D. H.; Lough, A. J. The acetyl derivative of humirianthol. Acta Crystallographica, Section E, 2003, E59, o347-o349.
25
OH
O O H H HO H H O O 3.4
Compound 3.5 was also isolated as a white solid and HRFABMS indicated a molecular
formula of C20H29O6. The major difference between the spectra of compound 3.5 and compound
3.4 was the absence of signals for the tetrahydrofuran ring in compound 3.5. The difference was
noticed in the 1H NMR spectrum where the C-16 proton resonated at δ 4.52 (dd, J= 9.5, 2.1 Hz) for compound 3.4 and at δ 4.29 (m) and 4.46 (m) for compound 3.5. The absence of the peak at δ
86.5 and the presence of one at δ 61.12 in the 13C NMR spectrum of compound 3.5 confirmed the absence of a tetrahydrofuran moiety in compound 3.5. Compound 3.5 was also a known compound, and its NMR spectra agreed with previously reported data (Table 3.1).34
OH
OH
O H HO H H O O 3.5
Compound 3.6 was also isolated as a white solid and HRFABMS indicated a molecular formula of C20H27O6. Like compound 3.5, the absence of the peak at δ 86.5 and the presence of
one at δ 64.13 in its 13C NMR spectrum confirmed the absence of the tetrahydrofuran moiety in
compound 3.6. Although compound 3.6 is a known compound, there was no published NMR
26 data of the compound.6,35 Its NMR data matched those of compound 3.5 closely, except for the
signals for the side chain atoms and the C-13 carbon.
O
OH
O H HO H H O O 3.6
Compound 3.7 was also isolated as a white solid and HRFABMS indicated a molecular
formula of C20H25O7. Its NMR spectra were almost identical to the spectra of compound 3.4
except for the absence of the singlet at δ 4.14 attributed to the C-14 proton in the spectrum of compound 3.4 and the presence of a peak at δ 106.36 in the 13C NMR spectrum. In the HMBC spectrum of compound 3.7, C-17 correlated to C-14 (δ 106.13) and C-14 also correlated to C-15.
The position of the hydroxyl group was determined based on these HMBC correlations.
Compound 3.7 was also known, but there was no published NMR data; its structure was established through X-ray crystallography.36
35 Mussini, P; Orsini, F; Pelizzoni, F.; Ferrari, G. Constituents of Annona coriacea. Structure of a new diterpenoid. J. Chem. Soc., Perkin Trans. 1 1973, 2551-7
36 On'okoko, P; Vanhaelen, M.; Vanhaelen-Fastre, R.; Declercq, J. P.; Van Meerssche, M. The constitution of icacinol, a new diterpene with a pimarane skeleton from Icacina claessensis. Tetrahedron 1985, 41, 745-8.
27 OH
O O H OH HO H H O O 3.7
Compound 3.8 was also isolated as a white solid and HRFABMS indicated a molecular
formula of C19H25O6. Its NMR spectrum was almost identical to the spectra of the other
compounds without the tetrahydrofuran ring. The 1H NMR spectrum exhibited a pair of doublets
at δ 3.63 (d, J = 8.9 Hz, 1H) and at δ 4.24 (d, J = 8.9 Hz, 1H) and also a doublet of doublets at δ
4.97 (dd, J = 6.5, 6.3 Hz, 1H). The remainder of the spectrum was almost identical to the spectra
of the other compounds. The 13C NMR spectrum had only two oxygenated carbon peaks at δ
72.83 and 73.03, and two carbonyl peaks at δ 179.74 and 179.5. Compound 8 was previously
obtained from the oxidation of annonalide with periodic acid,8 but was isolated here for the first time as a natural product.
O
OH
O H HO H O O 3.8
Compounds 3.1, 3.3, 3.4, 3.5, 3.6, and 3.7 exhibited cytotoxic activity against the A2780 human ovarian cancer cell line with IC50 values of 2.2, 0.8, 1.1, 1.4, 0.67 and 0.63 μg/mL
respectively. Compounds 3.2 and 3.8 were weakly active with IC50 values of 16 and 14 μg/mL,
respectively
28 1 13 Table 3.1: H and C NMR data in C5D5N for Compounds 3.1-3
Humirianthone (3.1) 1-Hydroxy-humirianthone (3.2) 15R-Humirianthol (3.3) Position δ H δ C δ H δ C δ H δ C 1 1.62 m 29.18 4.25 dd(1.9,3.9) 66.60 1.64 m 28.50 1.68 m 1.71 m 2 2.22 m 28.52 2.99 dd(4, 4) 41.52 2. 22 m 28.23 2.25 m 2.25 m 3 97.17 97.92 97.18 4 50.84 51.21 50.89 5 2.34 dd(2.6, 6.9) 45.12 2.70 dd (2.6, 7) 43.84 2.34 dd(2.9, 12.2) 45.31 6 4.98 dd(2.3, 4.8) 72.51 5.13 dd(2.3, 4.8) 71.78 4.96 dd(4.8, 6.8) 72.60 7 6.10 d(4.9) 119.22 6.16 (d 4.6) 119.52 5.96 d(4.8) 116.91 8 144.32 145.12 147.11 9 1.68 dd(12.1, 2.9) 37.11 2.41 dd(2.6, 7) 36.60 1.78 dd(12.2, 2.9) 37.66 10 30.69 30.12 30.79 11 1.18 m 25.55 1.18m 25.70 1.18 m 25.89 1.58 m 1.58 m 1.61 m 12 1.32 m 29.68 1.32 m 30.18 1.21 m 29.40 13 50.14 50.71 47.25 14 4.15 s 87.82 4.19 87.80 3.9 s 88.08 15 216.18 216.70 4.05 t(8.1) 79.36 4.23 t(8.1) 16 3.97 d(17.2) 70.70 3.96 d(16.95) 72.32 3.68 dd(1.9, 8.9) 72.68 4.53 d(17.2) 4.47 d(16.95) 4.33 dd(1.9, 8.9) 17 1.0 14.80 1.03 15.53 1.04 s 19.26 18 1.56 19.22 1.60 19.88 1.55 s 19.42 19 179.24 179.38 179.53 20 3.68 dd(3.4, 5.9) 72.12 4.33 dd(3.4, 5.9) 67.18 3.68 dd(1.9, 8.9) 71.91 4.31 dd(3.4, 5.9) 4.64 dd(3.4, 8.9) 4.33 dd(1.9, 8.9) 1-OH 6.90 s 6.87 d(J = 4.0) 3-OH 7.02 s 6.90 6.83 s 15-OH 6.82 d (4)
29 13 Table 3.2: C NMR data in DMSO-d6 for compounds 3.4-3.8
Carbon 3.4 Lit data33 3.5 Lit. data35 3.6 3.7 3.8 1 28.28 28.30 28.18 28.20 30. 29.47 29.14 2 27.94 27.96 27.94 27.96 26.38 28.53 28.47 3 96.13 96.16 96.13 96.16 96.57 97.17 97.19 4 49.79 49.80 50.01 50.02 50.34 50.90 51.01 5 43.78 43.80 43.36 43.38 43.47 44.81 44.68 6 71.02 71.05 72.14 72.17 72.51 72.59 73.15 7 116.38 116.38 113.66 113.66 115.6 116.54 115.59 8 145.56 145.58 46.36 146.47 144.50 149.64 146.02 9 36.20 36.21 42.37 42.38 42.66 39.12 42.91 10 29.78 29.80 29.88 29.91 30.71 30.88 30.78 11 24.91 24.90 24.31 24.30 24.17 25.91 24.83 12 31.88 31.91 34.00 34.03 33.47 34.60 35 13 48.09 48.10 39.51 39.52 48.37 50.87 45.16 14 85.26 85.26 43.89 43.89 43.37 106.37 42.91 15 77.43 77.41 79.38 79.41 213.56 79.07 179.70 16 75.06 75.08 61.98 62.00 64.13 75.72 17 15.03 15.04 17.81 17.82 18.98 14.19 19.15 18 18.29 18.30 18.30 18.30 19.35 19.22 19.15 19 178.16 178.17 78.42 178.43 180.16 179.2 179.1 20 71.03 71.04 71.81 71.82 72.51 72.71 72.82
3-3 Experimental Section
3.3.1 General Experimental Procedures
Optical rotations were measured with a Perkin-Elmer Model 241 polarimeter. IR and UV spectra were measured on MIDAC M-series FTIR and Shimadzu UV-1201 spectrophotometers, respectively. The NMR spectra were obtained on either a JEOL Eclipse 500 spectrometer. The mass spectra were obtained on a JEOL JMS-HX-110 instrument. A flash chromatograph from
Biotage Inc. was used for flash chromatography. HPLC was performed on a Shimadzu LC-10AT
30 instrument using a Varian Dynamax C18 column (250 ´ 10 mm). C-18 SPE columns were
obtained from Supelco.
3.3.2 Cytotoxicity Bioassays
The A2780 human ovarian cancer cell line cytotoxicity assay was performed by Jennifer
Schilling at Virginia Polytechnic Institute and State University as previously described above.
3.3.3 Plant Material
A liana was collected from the rainforest in Akisiamaw village, Suriname, under the vernacular name “apukutatai” in June 2000. MeOH and EtOAc extracts of dried plant material were received from Suriname as part of the ICBG program.
3.3.4 Extraction and Isolation
The MeOH and EtOAc extracts (4g) were partitioned between hexane and 60% aqueous
MeOH, then followed by partition between CH2Cl2 and 50% aqueous MeOH. All the fractions were evaporated to dryness and tested for their biological activity, and only the CH2Cl2 fraction was active. The active fraction was submitted to reversed phase C-18 column chromatography
(MeOH/H2O,1/1) followed by reversed phase HPLC using the same solvent system as mobile phase to produce the three new diterpenoids 1 (15mg), 2 (1.7 mg), 3 (1.5 mg), as well as the five known diterpenoids 4 (20 mg), 5 (10 mg), 6 (1.1mg), 7 (1.2 mg), and 8 (0.8 mg) as colorless amorphous solids.
31 Casimirella sp. Extract # 940412 M
1g IC50(µg/mL) = 13
Partition between 60%aq.MeOH and Hexane
Partition between 50%aq.MeOH/ CH2Cl2 Hexane frax 240mg CH Cl frax IC50 (µg/mL) < 20 2 2 MeOH frax. 322.4mg 373.6mg IC50 (µg/mL)= 4 IC50 (µg/mL) < 20 CH2Cl2( E + M)frax 444 mg
C18-gradient 50%MeOH/H2O to 100% MeOH
1 2 Yield: mg 120.2 280.3
IC50(µg/mL) = 3.5 < 20
C18-Flash chroma 50% MeOH/H2O
1-1 1-2 1-4 1-5 Yield: mg 45.7501020
IC50 = 16.7 1.1 15 NA
1-2-1 1-2-2 1-2-31-2-41-2-51-2-61-2-71-2-8 Yield: mg 14 13 6.5 11.3 2.2 6 1.1
IC50 (µg/mL) 0.67 1.41.1140.63 0.8 2.2 16
Figure 3.3 Bioassay guided fractionation of Casimirella sp.
32 -1 Compound 3.1: white amorphous solid (13 mg); IR nmax cm 3496 br, 2922, 1761 1663
-1 1 13 + cm , H and C NMR (Table 3-1). HRFABMS m/z found 361.1641 [M+H] ; calcd for C20H25O6,
20 361.1651; [α]D = -119°
-1 Compound 3.2: white amorphous solid (1.3 mg); IR nmax 3496 br, 1761 ,1663 cm ;1H
13 + and C NMR (Table 3-1); HRFABMS m/z found 377.1624 [M+H] ; calcd for C20H25O7,
20 377.1600 [α]D = -27°(CHCl3/MeOH 1/1, c 0.05)
-1 -1 1 Compound 3.3: white amorphous solid (1 mg); IR nmax cm 3550 br, 2940, 1761 cm H
13 + and C NMR (Table 3-1). HRFABMS m/z found 363.1824 [M+H] ; calcd for C20H27O6,
20 363.1808 [α]D = -91° (CHCl3/MeOH 1/1, c 0.06);
1 Compound 3.4: white amorphous solid (14 mg); H NMR (500 MHz, C5D5N) δ 1.20 (s),
1.55 (s), 1.18 (m), 1.21 (m), 1.61 (m), 1.64 (m), 1.72 (m), 2.22 (m), 2.25 (m), 2.34 (dd, J = 2.9,
12.2 Hz), 3.65 (dd, J =1.9, 8.9 Hz), 4.00 (s), 4.09 (d, J = 8.1 Hz), 4.33 (dd, J = 1.9, 8.9 Hz), 4.52
(dd, J = 4.8, 1.8 Hz), 6.71 (s), 6.81 (s), 8.83 (s). 13C NMR (Table 3.2) HRFABMS found m/z
+ 363.1824 [M+1] ; calcd for C20H27O6 363.1808
1 Compound 3.5: white amorphous solid (1.3 mg); H NMR (500 MHz, C5D5N) δ 1.31 (s),
1.54 (m), 1.55 (m), 1.58 (m), 1.76 (m), 1.92 (m), 1.93 (s), 2.14 (dd, J = 2.9, 12.2 Hz), 2.47 (m),
2.60 (m), 2.67 (dd, J = 11.2, 2.3 Hz), 3.96 (dd, J =2.1, 8.9 Hz), 4.12 (dd, J = 1.8, 8.1 Hz), 4.29
(m), 4.46 (m), 4.61 (dd, 1.8, 8.9 Hz),5.35 (dd, J = 5, 7 Hz), 6.05 (d, J = 4.2 Hz), 6.49 (d, J = 4.1
Hz), 6.62 (d, J = 4.0 Hz)), 7.09 (s); 13C NMR (Table 3.2); HR-FABMS m/z found 365.1960
+ [M+1] ; calcd for C20H29O6 365.1964
33 1 Compound 3.6: white amorphous solid (2.2 mg); H NMR (500 MHz, CDCl3) 1H NMR
δ 1.31 (s), 1.48 (m), 1.52 (m), 1.52 (m), 1.77 (m), 1.83 (m), 1.92 (s), 2.16 (dd, J = 2.9, 12.2 Hz),
2.48 (m), 2.60 (m), 2.68 (dd, J = 11.2, 2.3 Hz), 3.97 (dd, J =2.1, 8.9 Hz), 4.17 (dd, J = 1.8, 8.1
Hz), 4.29 (m), 4.46 (m), 4.61 (dd, 1.8, 8.9 Hz),5.35 (dd, J = 5, 7 Hz), 6.05 (d, J = 4.2 Hz), 6.49 (d,
J = 4.1 Hz), 6.62 (d, J = 4.0 Hz)), 7.09 (s). 13C NMR (Table 3.2). HRFABMS m/z found
+ 365.1961 [M+1] ; calcd for C20H29O6 365.1964
Compound 3.7: white amorphous solid (6 mg); 1H NMR (500 MHz, C5D5N) δ 1 (s),
1.55 (s), 1.18 (m), 1.21 (m), 1.61 (m), 1.64 (m), 1.72 (m), 2.22 (m), 2.25 (m), 2.34 (dd, J = 2.9,
12.2 Hz), 3.65 (dd, J =1.9, 8.9 Hz), 4.00 (s), 4.09 (d, J = 8.1 Hz), 4.33 (dd, J = 1.9, 8.9 Hz), 4.52
13 (dd, J = 4.8, 1.8 Hz), 8.63 (s, 3-OH), 6.71 (s), 6.81 (s). C NMR (500 MHz, C5D5N, Table 3-2).
+ HRFABMS, m/z found 377.1604 [M+1] ; calcd for C20H25O7 377.1600.
+ Compound 3.8: white amorphous solid (1.1 mg); HRFABMS C19H25O6 and m/z [M+1]
= 349.1624. 1H NMR (500 MHz, C5D5N) δ 1 (s), 1.55 (s), 1.18 (m), 1.21 (m), 1.61 (m), 1.64 (m),
1.72 (m), 2.22 (m), 2.25 (m), 2.34 (dd, J = 2.9, 12.2 Hz), 3.65 (dd, J =1.9, 8.9 Hz), 4.00 (s), 4.09
(d, J = 8.1 Hz), 4.33 (dd, J = 1.9, 8.9 Hz), 4.52 (dd, J = 4.8, 1.8 Hz), 8.63 (s, 3-OH), 6.71 (s), 6.81
13 + (s). C NMR (500 MHz, C5D5N) see Table 3-2. HR-FABMS m/z found 349.1624 [M+1] ; calcd for C19H25O6 349.1651.
34 IV. CARDENOLIDE GLYCOSIDES FROM PENTOPETIA ANDROSAEMIFOLIA
4.1 Introduction
Pentopetia androsaemifolia Decne belongs to the family Asclepiadaceae. No previous
chemical studies of this plant have been reported. Roots, stems and leaves of Pentopetia
androsaemifolia were collected in Madagascar as a part of the ICBG program in order to
investigate their potential anticancer activity. Only extracts of the roots and stems were active
against the A2780 cell line.
4.2 Results and Discussion
The EtOH extract of the roots and stems of Pentopetia androsaemifolia was subjected to
the solvent partitioning between hexane and aqueous MeOH, and the aqueous MeOH was then
extracted with CH2Cl2. All the phases were evaporated under reduced pressure and tested for
their biological activities against the A2780 ovarian cancer cell line. The CH2Cl2 and MeOH
fractions were both active fractions. The MeOH fraction was then partitioned between BuOH
and water, and both fractions tested for their activity. The BuOH fraction was the only active
fractions. Thin layer chromatography (TLC) revealed that both the CH2Cl2 and BuOH active
fractions contained similar constituents. Therefore they were combined and purified through
reversed phase preparative HPLC using MeOH/H2O (6/4) as mobile phase to yield three known cardenolide glycosides (4.1, 4.2, and 4.4) and one new cardenolide glycoside 4.3.
Compound 4.1 was isolated as a white amorphous powder, and HRFABMS indicated a
1 molecular formula of C42H66O18. Its H NMR spectrum revealed two singlets at δ 0.88 (s, 3H,
H3-18), and 0.92 (s, 3H, H3-19). Diagnostic peaks were also observed at δ 2.83 (dd, 1H, J = 9.0,
6.0 Hz, H-17), δ 4.91 (dd, 1H, J = 19, 1.3 Hz, H-21), δ 5.02 (dd, 1H, J = 19, 1.2 Hz, H-21) and δ
35 5.89 (brs, 1H, H-22). These peaks, coupled with the two singlets (2 x CH3), suggested that
compound 4.1 could be a cardenolide, since these chemical shifts are characteristic of the aglycon portion of cardenolides. The 13C NMR spectrum of compound 4.1 showed two peaks at
δ 177.24 for C-23 and at δ 178.37 for C-20; these signals indicative are of α,β-unsaturated γ-
lactone unit of cardenolides. The 1H NMR spectrum had signals for three anomeric protons at δ
4.35 (d, J = 8 Hz, 1H), δ 4.34 (d, J = 7.8 Hz, 1H) and δ 4.95 (dd, J = 9.4, 2.0 Hz, 1H) indicating the presence of three sugars. This conclusion was confirmed by its 13C NMR spectrum, which contained signals for three anomeric carbons at δ 98.19, 105.20, and 106.23. The presence of a doublet at δ 1.28 (d, J = 6 Hz, 3H), a doublet of doublets of doublets at δ 2.15 (ddd, J = 14.0, 3.2,
2.1 Hz) and a singlet at δ 3.45 (s, 3H) representing one methoxy group indicated that one of the sugars could be cymarose. The final structure and the connectivity of the three sugars were confirmed by the COSY and HMBC correlations. The stereochemistries of the sugars was determined using ROESY, 1D-TOCSY and by calculation of the different coupling constants of the sugars. The NMR data for compound 4.1 combined with its elemental composition indicated that it was most probably a cardenolide glycoside of the periplogenin class previous isolated from Biondia hemsleyana. A comparison of its NMR data with the literature data of this glycoside37 indicated that compound 4.1 is periplogenin-3-O-[β-D-glucopyranosyl-(1→6)-β-D- glucopyranosyl-(1→4)-β-D-cymaropyranoside] (Table 4-1). It had an IC50 of 0.22 µg/mL against
the A2780 cell line.
37 Tan, X G.; Zhang, X., R.; Wang, M. K.; Peng, S. L. Two new cardenolide glycosides from Biondia hemsleyana. Chinese Chemical Letter, 2002, 13, 547-548
36 O
O OH O HO HO O OH H3C OH O O HO O O HO OH OH OCH3 4.1
Compound 4.2 was also isolated as a white amorphous powder and its HRFABMS
indicated a molecular formula of C36H56O13. Its NMR data were similar to those of compound
4.1, except that it had signals for only two anomeric protons at δ 4.33 (d, J = 8.0 Hz, 1H) and δ
4.95 (dd, J = 9.4, 2.0 Hz, 1H) in its 1H NMR spectrum, and for only two anomeric carbons at δ
98.14 and 106.23 in the 13C NMR spectrum. These observations suggested that compound 4.2
had two sugars. The final structure and the connectivity of the two sugars were also confirmed
by COSY and HMQC correlations and the HMBC correlations. The stereochemistries of the
sugars were determined using ROESY, 1D-TOCSY and by measuring proton-proton coupling
constants of the sugars. The NMR data for compound 4.2, combined with its elemental
composition indicated that it was most probably a cardenolide glycoside of the periplogenin class.
A comparison of its NMR data with the literature data of periplogenin-β-D-glucopyranosyl-β-D- cymaropyranoside]. 38 indicated that compound 4.2 was the known periplogenin-3-O-[β-D- glucopyranosyl-(1→4)-β-D-cymaropyranoside]. It had an IC50 of 0.24 µg/mL against the A2780 cell line.
38 Junping, X.; Koichi, T.; Hideji, I.. Pregnanes and cardenolides from Periploca sepium. Phytochemistry, 1990, 29, 244-246
37 O
O
OH H3C OH O O HO O O HO OH OH OCH3 4.2
Compound 4.3 was also isolated as a white amorphous powder and its HRFABMS
1 indicated a molecular formula of C43H68O16. Its H NMR spectrum in C5H5N revealed two
singlets at δ 0.86 (s, 3H, H3-19), and 0.98 (s, 3H, H3-18), and additional diagnostic peaks at δ
2.75 (dd, 1H, J = 9.0, 6.0 Hz, H-17), δ 4.98 (dd, 1H, J = 19.7, 1.6 Hz, H-21), δ 5.30 (dd, 1H, J =
19.7, 1.2 Hz, H-21) and δ 6.02 (brs, 1H, H-22). These peaks taken together suggested that
compound 4.3 could also be a cardenolide. The aglycone of this cardenolide lacked a hydroxyl
group at the C-5 position, based on its HMBC correlations (Figure 4.1). Its NMR data were
obtained in both CD3OD and C5H5N. In C5H5N, the signals for protons H3-18 and H3-19
switched position. Its 13C NMR spectrum also showed signals at δ 174.52 for C-23 and at δ
175.93 for C-20 which are indicative of the α,β-unsaturated γ-lactone unit of cardenolides.
Its 1H NMR spectrum had signals for three anomeric protons at δ 4.65 (d, H-1″, J = 7.8
Hz, 1H), δ 5.07 (d, H-1′″ J = 7.8 Hz, 1H) and δ 5.16 (dd, H-1′, J = 9.4, 2.0 Hz, 1H) indicating the presence of three sugars. This conclusion was confirmed by its 13C NMR spectrum, which had signals for three anomeric carbons at δ 96.61 (C-1′), 105.65 (C-1′″), and 106.65 (C-1″). The 1H
NMR spectrum also showed a doublet at δ 1.56 (d, H-6′″ J = 6.2 Hz, 3H), a doublet at δ 1.62 (d,
H-6′, 6.2 Hz, 3H), a doublet of doublets of doublets at δ 2.30 (ddd, J = 14.0, 3.2, 2.1 Hz) and two singlets at δ 3.41 (s, 3H) and 3.63 (s, 3H) for two methoxy groups. The final structure and the
38 connectivity of the sugars were confirmed by its COSY and HMQC spectra and by HMBC correlations depicted in Figure 4-1. The stereochemistry of the sugars was determined using
ROESY, 1D-TOCSY and by comparison of proton-proton coupling constants with those previously determined for β-D-glycosyl-β-D-digitaloside 39 and the 13C NMR chemical shifts of the sugar moiety part b to those of the cannogenin-β-D-glycosyl-β-D-digitaloside isolated from
Apocynum cannabinum.39 Part a of the sugar moiety is identical to the same unit in compounds
4.1 and 4.2 (Table 4-3). The 13C NMR chemical shifts of the aglycone part were identical to those of digitoxigenin 3-O-[β-D-glucopyranosyl-(1→4)-2′-O-acetyl-α-L-thevetopyranoside. 40
The NMR data for compound 4.3 combined with its elemental composition indicated that it was a cardenolide glycoside of the digitoxigenin class. It was determined to be the new cardenolide glycoside, digitoxigenin-β-D-glycosyl-β-D-digitaloside-β-D-cymaropyranoside. It had an IC50 of
0.22 µg/mL against the A2780 cell line.
O
O HOH H O HO O HO H OH H C 3 H3C OH H H H O O H O O H3CO H H OH H H H OCH3 H 4.3 Part b Part a
39 Abe, F.; Yamauchi, T. Cardenolide glycoside from the roots of Apocynum cannabinum. Chem.Pharm.Bull. 1994, 42, 2028-2031 40 Endo,H.; Warashina, T.; Noro, T. ; Castro, V. H.; Mora, G. A., Poveda, L. J.; Sanchez, P. E. Cardenolides from Thevetia ahouai (Linn.) A.DC. Chem. Pharm. Bull. 1997, 45, 1536-1538
39 O
O HOH H O HO HO O H H C OH 3 H3C OH H H H O O H O O H3CO H H OH H H H OCH H 4.3 3
Figure 4.1 HMBC correlations of compound 4.3
Table 4.1 1H NMR data of the sugar moiety of compound 4.3
Sugar I 1′ 5.07 (d, 1H, J = 7.8 Hz, H-1’) 2′ 2.30 (ddd, 2H, J = 14.2, 3.2, 2.1 Hz, H2-2’), 1.90 (ddd, 2H, J = 12.5, 12.0, 9.5 Hz, H2-2’) 3′ 4.04 (brd, 1H, J = 3 Hz, H-3’) 4′ 3.61 (br d, 1H, J = 5.0 Hz, H-4’) 5′ 4.12 (dq, 1H, J = 10, 6.0 Hz, H-5’) 6′ 1.61 (d, 3H, J = 6 Hz, H-6’) 3′-O-CH3 3.41 (s, 3H) Sugar II 1″ 4.65 (d, 1H, J = 7.8 Hz, H-1”) 2″ 4.37 (m, 1H, H-2”) 3″ 3.50 (dd, 1H J = 9.9, 3.2 Hz, H-3”) 4″ 4.25 (dd, 1H, J = 4.5, 2.5 Hz, H-4”) 5″ 3.69 (d, 1H, J = 6.4 Hz, H-5”) 6″ 1.54 (d, 3H, J = 6.2 Hz, H-6”) 3″-OCH3 3.64 (s, 3H) Sugar III 1″′ 5.16 (d, 1H, J = 9.4 Hz, H-1”’) 2″′ 3.92 (t, 1H, J = 7.8 Hz, H-2”’), 3″′ 4.18 (t, 1H, J = 8 Hz, H-3”’) 4″′ 3.93 (m, 1H, H-4”’) 5″′ 4.08 (m, 1H, H-5”’), 6″′ 4.56 (br d, 2H, J = 10.2 Hz, H-6”’), 4.25(br d, 2H, J = 10 Hz, H-6”’)
Compound 4.4 was also isolated as a white amorphous substance. Its HRFABMS
1 indicated a molecular formula of C30H35O8. Its H NMR spectrum revealed two singlets at δ 0.88
40 (s 3H, H3-18) and δ 0.94 (s, 3H, H3-19), a doublet at δ 1.27 (d, 3H, J = 6.1, H3-6′) and a doublet of doublets at δ 2.78 (dd, 1H, J = 9.3, 5.6, H-17). It had one methoxy group at δ 3.43 (s, 3H), and one anomeric proton at δ 4.78 (dd, 1H, J = 9.9, 2.1, H-1′). The presence of a single sugar was confirmed by its 13C NMR and by signal at δ 96.44, two doublets of doublets at δ 4.98 (dd, 1H, J
= 18.2, 1.3 Hz, H-21) and δ 4.81 (dd, 1H, J = 18.2, 1.8 Hz, H-21), and a singlet at δ 5.88 (brs, 1H,
H-22) in its 1H NMR spectrum. Based on its 13C NMR data and its HMBC correlations, compound 4.4 had the same aglycone as compounds 4.1 and 4.2. The NMR data for compound
4.4 combined with its elemental composition indicated that it was most probably a cardenolide glycoside of the periplogenin class. A comparison of its NMR data with the literature data of the cardenolide glycoside periplocymarin 41 indicated that compound 4.4 was periplocymarin. It had an IC50 of 0.4 µg/mL against the A2780 cell line.
O O
H3C OH O HO O OH H OCH3 H 4.4
41 Ueda, J.; Tezuka, Y.; Banskota, A. H.; Tran, Q. L.; Tran, K. Q.; Saiki, I.; Kadota, S. Constituents of the the Vietnamese medicinal plant Streptocaulon juventas and their antiproliferative activity against the HT-1080 fibrosarcoma cell. J. Nat. Prod. 2003, 66, 1427-1433
41 Table 4.2 13C NMR spectra of compounds 4.1, 4.2, 4.4
4.1a Lit data37 4.2a,b Lit. data38 4.4c Lit.data41 Aglycon 1 25.8 25.6 25.9 24.9 25.4 25.4 2 26.2 26.0 26.3 25.3 26.2 26.1 3 75.1 74.7 75.3 74.9 75.3 75.3 4 35.2 34.9 35.2 34.3 34.2 34.6 5 73.5 73.5 73.6 72.7 73.5 73.6 6 35.1 34.9 35.4 34.3 33.8 34.1 7 24.2 23.9 24.2 23.3 23.7 23.6 8 40.8 40.6 40.8 39.9 40.9 40.8 9 39.1 38.8 39.1 38.1 40.1 39.2 10 41.1 40.8 41.1 40.1 40.7 40.7 11 21.9 21.7 21.9 20.9 21.6 21.5 12 39.8 39.6 39.8 38.8 39.20 40.1 13 49.9 49.7 49.8 48.9 49.4 49.4 14 84.5 84.4 84.5 83.6 85.6 85.5 15 32.9 32.7 33.0 32.01 33.0 33.0 16 27.1 26.9 27.1 26.2 26.8 26.8 17 51.1 50.9 51.2 50.2 50.7 50.7 18 16.0 15.9 16.0 15.1 15.7 15.7 19 17.1 16.8 17.1 16.1 16.8 16.7 20 175.8 175.9 175.8 174.9 174.2 174.5 21 73.5 73.5 73.6 72.7 73.4 73.4 22 117.6 117.2 117.7 116.6 117.8 117.7 23 174.5 174.5 174.5 173.5 174.3 174.4 Sugar I 1′ 97.3 97.0 97.2 96.3 96.4 96.4 2′ 36.5 36.1 36.3 35.5 34.6 33.8 3′ 77.9 77.8 78.3 77.2 77.3 77.3 4′ 83.2 82.8 82.8 81.8 72.3 72.3 5′ 69.5 69.2 69.4 68.4 70.9 70.9 6′ 18.5 18.3 18.6 17.6 18.2 18.2 3′-OCH3 58.5 58.3 58.4 57.5 57.4 57.3 Sugar II 1″ 105.6 105.1 106.5 105.3 2″ 75.2 74.7 75.3 74.2 3″ 77.9 77.9 78.4 77.7 4″ 71.8 71.3 71.7 70.7 5″ 76.8 76.5 77.8 76.8 6″ 70.3 70.3 62.9 61.9 Sugar III 1″′ 106.5 106.0 2″′ 75.9 75.4 3″′ 78.4 78.0 4″′ 71.6 71.4 5″′ 78.5 78.0 6″′ 62.7 62.3 a) δ values in C5D5N; b) The literature data were obtained using TMS as reference, while the experimental data were obtained using the C-4 carbon of pyridine at 135.91 ppm as the reference. Since the chemical shift of this carbon is sensitive to conditions has been cited as high as 138.7 ppm (Crews, P.; Rodriguez , J.; Jaspars, M.; Organic Structure Analysis Oxford University Press, New York, 1998, p 88), the systematic deviation of the experimental results from the literature data by about 1.0 ppm is most probably due to a variation in the chemical shift of the pyridine reference signal. c) δ in CDCl3.
42 Table 4.3 13C NMR and 1H NMR spectra of compound 4.3
δ Carbon a Lit.data of aglycone part39,a Aglycon 1 30.8 29.9 2 27.3 27.0 3 73.4 72.9 4 30.6 31.0 5 37.0 37.0 6 27.0 27.2 7 21.5 21.6 8 41.9 42.0 9 35.8 35.8 10 35.5 35.6 11 22.0 22.0 12 39.8 39.9 13 50.1 50.2 14 84.6 84.6 15 33.1 33.2 16 27.0 27.4 17 51.4 51.5 18 16.2 16.2 19 23.8 24.2 20 176.0 175.9 21 73.7 73.7 22 117.6 117.7 23 174.5 174.5 Sugar I Sugar I compared to sugar I of 4.137,a 1′ 96.6 97.0 2′ 36.8 36.1 3′ 77.9 77.8 4′ 83.6 82.8 5′ 69.4 69.2 6′ 18.8 18.3 3′-O-CH3 58.3 58.3 Sugar II Sugar II and III compared to β-D-glucosyl- β-D-digitaloside39,a 1″ 106.6 103.4 2″ 71.4 71.2 3″ 85.3 85.5 4″ 76.7 76.6 5″ 70.6 70.5 6″ 17.6 17.7 3″-OCH3 59.0 58.9 Sugar III 1″′ 105.6 105.4 2′″ 75.9 76.0 3′″ 78.3 78.5 4″′ 71.7 71.9 5′″ 78.6 78.3 6″′ 63.1 63.1 a) In C5D5N
43 4.3 Experimental Section
4.3.1 General Experimental Procedure
Optical rotations were measured with a Perkin-Elmer Model 241 polarimeter. The NMR spectra were obtained on either a JEOL Eclipse 500 spectrometer or on a Inova 400 spectrometer.
The mass spectra were obtained on a JEOL JMS-HX-110 instrument. A flash chromatograph from Biotage Inc. was used for flash chromatography. HPLC was performed on a Shimadzu LC-
10AT instrument using a Varian Dynamax C18 column (250 ´ 10 mm). C-18 SPE columns were
obtained from Supelco.
4.3.2 Cytotoxicity Bioassays
The A2780 human ovarian cancer cell line cytotoxicity assay was performed at Virginia
Polytechnic Institute and State University as previously described above.
4.3.3 Plant Material
The extracts, root, stem and leaves of Pentopetia androsaemifolia Decne were collected by F. Ratovoson and his assistants from the rainforest of Toamasina, Madagascar, in the vicinity of Vohimena on December 08, 2001. The plant was identified by F. Ratovoson. The roots and stem of the dried plant material were extracted with EtOH to yield extracts MG 1228 and 1229
4.3.4 Extraction and Isolation
Extracts MG 1228 and 1229 from Pentopetia androsaemifolia (2 g) were partitioned
between hexane and 60% aqueous MeOH, and the latter extract was diluted to 50% aqueous
MeOH and extracted with CH2Cl2. All the resulting fractions were evaporated to dryness and
44 tested for their biological activity. The CH2Cl2 and MeOH fractions were the most active with
IC50 values of 0.4 and 0.4 µg/mL. The MeOH fraction was then partitioned between BuOH and water, and tested for their activity. The BuOH fraction was the only active fraction. Thin layer chromatography (TLC) revealed that both the CH2Cl2 and BuOH fractions contained almost the
same constituents. Therefore they were combined and purified through reversed phase
preparative HPLC to yield compounds 4.1-4.4
1 Compound 4.1: white amorphous powder (15 mg); H NMR (500 MHz, CD3OD)
selected chemical shifts; δ 0.88 (s, 3H, H3-18), 0.92 (s, 3H, H3-19), 2.83 (dd, 1H, J = 9.0, 6.0 Hz,
H-17), 4.91 (dd, 1H, J = 19, 1.3 Hz, H-21), 5.02 (dd, 1H, J = 19, 1.2 Hz, H-21), 5.89 (brs, 1H, H-
13 22). C NMR (500 MHz, C5D5N) (Table 4.1); HRFABMS m/z found 881.4145 [M + Na]; calcd
for C42H66NaO18 881.4147
1 Compound 4.2: white amorphous powder (8 mg); H NMR (500 MHz, CD3OD);
selected chemical shift δ 0.87 (s, 3H, H3-18), 0.93 (s, 3H, H3-19).δ 2.83 (dd, 1H, J = 9.0, 6.0 Hz,
H-17), 4.33 (dd, J = 9.4, 2.0 Hz, 1′-H), 4.92 (dd, 1H, J = 19, 1.3 Hz, H-21), 5.02 (dd, 1H, J = 19,
13 1.2 Hz, H-21), 5.89 (brs, 1H, H-22). C NMR (500 MHz, C5D5N), (Table 4.1); HRFABMS m/z found 719.3614 [M + Na]; calcd for C36H56NaO13 719.3618
1 Compound 4.3: white amorphous powder (7.5 mg); H NMR (500 MHz, C5H5N)
selected chemical shifts; δ 0.86 (s, 3H, H3-19), and 0.98 (s, 3H, H3-18), 2.75 (dd, 1H, J = 9.0, 6.0
Hz, H-17), 4.98 (dd, 1H, J = 19.7, 1.6 Hz, H-21), 5.30 (dd, 1H, J = 19.7, 1.2 Hz, H-21), 6.02 (br s, 1H, H-22). Sugar moiety: δ 5.07 (d, 1H, J = 7.8 Hz, H-1’), 2.30 (ddd, 2H, J = 14.2, 3.2, 2.1 Hz,
45 H2-2’), 1.90 (ddd, 2H, J = 12.5, 12.0, 9.5 Hz, H2-2’), 4.04 (brd, 1H, J = 3 Hz, H-3’), 3.61 (br d,
1H, J = 5.0 Hz, H-4’), 4.12 (dq, 1H, J = 10, 6.0 Hz, H-5’), 1.61 (d, 3H, J = 6 Hz, H-6’), 3.41 (s,
3H, 3’-O-CH3), 4.65 (d, 1H, J = 7.8 Hz, H-1”), 4.37 (m, 1H, H-2”), 3.50 (dd, 1H J = 9.9, 3.2 Hz,
H-3”), 4.25 (dd, 1H, J = 4.5, 2.5 Hz, H-4”), 3.69 (d, 1H, J = 6.4 Hz, H-5”), 1.54 (d, 3H, J = 6.2
Hz, H-6”), 3.64 (s, 3H, 3”-O-CH3), 5.16 (d, 1H, J = 9.4 Hz, H-1”’), 3.92 (t, 1H, J = 7.8 Hz, H-
2”’), 4.18 (t, 1H, J = 8 Hz, H-3”’), 3.93 (m, 1H, H-4”’), 4.08 (m, 1H, H-5”’), 4.56 (br d, 2H, J =
13 10.2 Hz, H-6”’), 4.25(br d, 2H, J = 10 Hz, H-6”’). C NMR (C5D5N, Table 4.2); HRFABMS m/z found 863.4401 [M + Na]; calcd for C43H68NaO16 863.4405.
Compound 4.4: white amorphous powder (15 mg); 1H NMR 1H NMR (1H NMR (500
MHz, CDCl3) selected chemical shifts CDCl3; δ 0.88 (s, 3H, H3-18), 0.94 (s, 3H, H3-19), 2.78 (s,
1H, J = 9.3, 5.6 Hz, H-17), 4.98 (dd, 1H, J = 18.2, 1.3 Hz, H-21), δ 4.81 (dd, 1H, J = 18.2, 1.8
Hz, H-21), 5.88 (brs, 1H, H-22). Sugar moiety δ 4.78 (dd, 1H, J = 9.9, 2.1 Hz, H-1’), 2.23 (ddd,
2H, J = 14.2, 3.2, 2.1 Hz, H2-2’), 1.68 (ddd, 2H, J = 12.5, 12.0, 9.5 Hz, H2-2’), 3.63 (q, 1H, J =
3.2 Hz, H-3’), 3.22 (dd, 1H, J = 9.6, 3.2 Hz, H-4’), 3.63 (dq, 1H, J = 9.6, 6.0 Hz, H-5’), 1.27 (d,
13 3H, J = 6.1, H3-6’), 3.41 (s, 3H, 3-O-CH3). C NMR (CDCl3); (Table 4.1).HRFABMS m/z
+ found 535.3268 [M+H] ; calcd for C30H47O8 535.3271
46 Pentopetia androsaemifolia ( Asclepiadaceae) MG1228/1229
IC50 = 1.1 2 g
partition hexane/60% aqueous MeOH
partition CH2Cl2/50% aqueous MeOH
Hexane
Yield 152 mg CH2Cl2 MeOH % 7.6 Yield 207 mg1.63 g IC50 = > 20 % 10.4 81.3
IC50 = 0.4 0.4
1.8 g
258 mg used
R. phase chr.MeOH/H2O(7/3) followed by HPLC
12 3 4 5 6 Yield: 30 mg 15 mg 8 mg7.5 mg 15 mg 150 mg
IC50 = > 20 0.21 0.22 0.260.4 >20
Figure 4.2: Bioassay guided fractionation of Pentopetia androsaemifolia Decne
47 V. PHYSALINS FROM PHYSALIS ANGULATA L. (SOLANACEAE)
5.1 Introduction
The extract E/M 980288 was received from Suriname in 1999. This extract was initially believed to be of the plant Ertela trifolia, and subsequent testing for antimalarial activity at
Watter Reed Army Institute of Research indicated that it had good activity, with an IC50 of 11
µg/mL against Plasmodium falciparum. The plant Ertela trifolia had not previously been studied phytochemically and so it was selected for fractionation to isolate potential antimalarial compounds. Fractionation of E/M 980288 was guided by antimalarial bioassays conducted by a sister ICBG program in Panama, and led to the isolation of the seven compounds 5.1-5.7.
Structural studies of these compounds identified them all as known compounds of the physalin class, and this suggested that the plant might have been misidentified. Correspondence with our collaborators in Suriname confirmed this, and the plant was reidentified as Physalis angulata L
The plant Physalis angulata L. belongs to the Solanaceae family. It is a plant that has a worldwide distribution, from Africa through Asia, Mexico, and Central and South America. The plant is used in many developing countries to treat diseases as malaria, asthma, hepatitis, bacterial infections, and dermatitis, and many scientific papers have documented its biological properties. The plant has also been investigated phytochemically, and various compounds have been isolated from it. The roots, stems, twigs, fruits and leaves of Physalis angulata L. were collected in Suriname as a part of the ICBG program in order to investigate their potential antimalarial activity. The crude extract was originally tested for antimalarial activity at the
Walter Reed Army Institute of Research and at the “Instituto de Medicina Tropical y Ciencias de la Salud” in Panama and it had an IC50 of 11 µg/mL against Plasmodium falciparum in both assays.
48 5.2 Results and Discussion
The MeOH and EtOAc extracts from a mixture of leaves, twigs, fruits, roots and stems of
Physalis angulata L.(Solanaceae) were subjected to the usual partition between hexane and
aqueous MeOH, and the aqueous MeOH fraction was then extracted with CH2Cl2. All the fractions were evaporated under reduce pressure and tested for their biological activities against
Plasmodium falciparum, and only the CH2Cl2 fraction was active, with an IC50 of 11 µg/mL. The
active fraction was subjected to normal phase preparative HPLC using a gradient of
CH2Cl2/CH3CN (5% to 100% CH3CN) as mobile phase, and as a result seven known physalins
(5.1-5.7) were isolated.
Compound 5.1 was isolated as a white amorphous powder, and its HRFABMS indicated
1 a molecular formula of C28H30O9. Its H NMR revealed three singlets at δ 1.10 (s, 3H, H3-19),
and 1.15 (s, 3H, H3-28) and 1.76 (s, 3H, H3-21). The spectrum also showed a doublet of doublets
at δ 2.89 (dd, 1H, J = 20, 5 Hz, Hα-4), a doublet at δ 3.27 (br d, 1H, J = 20 Hz, Hβ-4), a doublet at
δ 3.58 (d, 2H, J = 12.1 Hz, H2-27), a doublet of doublets at δ 4.25 (dd, 2H, J = 14, 4.5 Hz, H2-27)
and also a doublet of doublets at δ 4.56 (dd, 2H, J = 3, 2 Hz, H-22). The spectrum also revealed
the presence of signals for two double bonds at δ 5.58 (br d, 1H, J = 6.2 Hz, H-6), 5.80 (dd, 1H, J
= 10, 2.3 Hz, H-2) and 6.89 (ddd, 1H, J = 10, 4.8, 2.2 Hz, H-3). The 13C NMR spectrum
indicated the presence of 28 carbons, including two lactone carbonyl signals at δ 167.3 and 171.8
assigned to C-26 and C-18 respectively and two ketone signals at δ 202.4 and 209.4 were
assigned to C-1 and C-15 respectively. Signals for five oxygenated carbons at δ 60.6, 76.3, 78.2,
80.3, 80.7 were assigned to C-27, C-22, C-13, C-20, and C-17 respectively and a signal for a
ketal carbon at δ106.3 was assigned to C-14. The 13C NMR spectrum also confirmed the
presence of two double bonds at δ 123.4, 126.9, 135.6, and 146.2 assigned to C-6, C-5, C-2 and
49 C-3 respectively. The NMR data for compound 5.1 combined with its elemental composition
indicated that it was most probably physalin B, previously isolated from the same plant. A
comparison of its NMR data with the literature data of physalin B indicated that compound 5.1
was indeed physalin B42 (Table 5.1).
H O O O O
O HO H O O O
5.1
Compound 5.2 was also isolated as a white amorphous powder, and its HRFABMS also
1 indicated a molecular formula of C28H30O9. Like that of compound 5.1, the H NMR spectrum of compound 5.2 showed the presence of three methyl groups at δ 1.14 (s, 3H, H3-19), 1.19 (s, 3H,
H3-28) and 1.78 (s, 3H, H3-21). Unlike compound 5.1, compound 5.2 had a doublet of doublets at δ 2.61 (dd, 1H, J = 19.9, 4.8 Hz, Hα-2), a doublet at δ 3.4 (br d, 1H, J = 20.2, Hβ-2), a multiplet at δ 5.63 (m, 1H, H-3), a broad doublet at δ 5.67 (br d, 1H, J = 2.8 Hz, H-6) and a doublet of doublets at δ 6.05 (dd, 1H, J = 9.8, 1.6 Hz, H-4). These signals in the 1H NMR spectrum of compound 5.2 meant that there was no a double bond conjugated to the C-1 carbonyl. The 13C
NMR spectrum of compound 5.2 confirmed the difference between the two compounds. This spectrum exhibited the presence of the same five oxygenated carbons at δ 60.7, 76.3, 78.3, 80.3 and 80.7, a ketal at δ 106.4, two carbonyl peaks at δ 167.2 and 171.7, and a ketone peak at 209.8; all these signals were assigned to the same carbons as in compound 5.1. The spectrum also
42 Januàrio, A. H.; Filho, E. R.; Pietro, R. C. L. R.; Kashima. S.; Sato, D. N.; França, S. C. Antimycobacterial physalins from Physalis angulata L. (Solanaceae). Phytother. Res. 2002, 16, 445-448
50 revealed the presence of two double bonds, but with different chemical shifts than those of
compound 5.1. The difference between the two compounds was thus between C-1, C-2, C-3, C-4
and C-5. The second carbonyl group in compound 5.2 resonated at δ 209.2 (C-1) while the C-1
carbonyl resonated at δ 202.4 in compound 5.1. The C-2 in compound 5.2 is a methylene group
and appeared at δ 39.6 which indicated that there was no a double bond conjugated to C-1 the
carbonyl. The 13C NMR spectrum also indicated the presence of two double bonds at δ 122.5 (C-
3), 125.8 (C-4), 140.4 (C-5) and 128.1 (C-6). The NMR data for compound 5.2 combined with its elemental composition, indicated that it was most probably the previous reported isophysalin
B.43 A comparison of its NMR data with the literature data of isophysalin B43 confirmed the identity of the the two compounds (Table 5-1). It should be noted that the literature 13C NMR
data of isophysalin B differ from these of compound 5.2 in the data for C-15, C-19 and C-28.
Since our data are fully consistent with the literature data for other similar compounds,42 it is
believed that the literature data for these resonances of isophysalin B are incorrectly recorded.
H O O O O
O HO H O O O
5.2
Compound 5.3 was also isolated as a white amorphous powder, and its HRFABMS also
1 indicated a molecular formula of C28H30O10. Its H NMR was similar to the two previous
compounds except, that it showed a doublet of doublets at δ 4.48 (dd, 1H, J = 3, 2 Hz, H-6)
43 Sunayama, R.; Kuroyanagi, M.; Umehara, K.; Ueno, A. Physalin and neophysalins from Physalis alkekengi var. francheti and their differentiation inducing activity. Phytochemistry. 1993, 34, 529-533
51 corresponding to a 6α-equatorial hydroxyl group, a doublet at δ 5.93 (d, 1H, J = 9.5 Hz, H-2), a
doublet at δ 6.17 (d, 1H, J = 6.5 Hz, H-4), and a doublet of doublets at δ 7.03 (dd, 1H, J = 11, 7
Hz, H-3). These signals indicated the presence of a 2,4-dienone system. Its 13C NMR spectrum
showed the presence of six oxygenated carbons at δ 60.7, 71.7, 76.3, 78.2, 80.4 and 80.8
assigned to C-27, C-6, C-22, C-13, C-20, and C-17 respectively, the usual ketal signal at δ 106.5,
lactone carbonyl peaks at 167.2 and 171.7, and also two ketone peaks at δ 205.1 (C-1) and 209.8
(C-15). It revealed also the presence of four olefinic carbons with signals at δ 116.5, 125.9, 140.0 and 158.1 assigned to C-4, C-2, C-3 and C-5 respectively. The NMR data for compound 5.3 combined with its elemental composition indicated that it was most probably the previously reported physalin G.44 A comparison of its NMR data with the literature data of physalin G44 indicated that compound 5.3 was indeed physalin G (Table 5.1).
H O O O O
O HO H O O O
OH 5.3
Compound 5.4 was also isolated as a white amorphous powder and its HRFABMS
1 indicated a molecular formula of C28H32O11. Its H NMR spectrum was identical to those of
compounds 5.1-5.3 except that its showed a doublet of doublets at δ 3.49 (brd, 1H, J = 3, 2 Hz
H-6) corresponding to a 6β-equatorial hydroxylated carbon, a doublet at δ 5.70 (dd, 1H, J = 10, 2
Hz, H-2), a doublet at δ 6.62 (ddd, 1H, J = 10, 5, 2 Hz, H-3), and a doublet of doublets at δ 1.98
44 Row, L, R. ; Reddy, K. S.; Sarma, S.; Matsuura, T.; Nakaashima, R. New physatin s from physalis angulata and physalis lancifolia. Structure and reactios of physalin D, I, G AND K; Phytochem. 1980, 19, 1175-1181
52 (dd, 1H, J = 20, 5 Hz, H-4α) and a broad doublet at δ 3.11 (br d, 1H, J = 20 Hz, H-4β). Its 13C
NMR spectrum showed the presence of seven oxygenated carbons at δ 60.5, 72.6, 76.3, 76.4,
78.7, 80.5 and 80.7 assigned to C-27, C-6, C-5, C-22, C-13, C-20, and C-17 respectively, the usual ketal signal at δ 106.9 and two lactone carbonyl peaks at 167.3 and 171.9, and also two ketone peaks at δ 204.0 (C-1) and 210.0 (C-15). It revealed also the presence of two olefinic carbons at 127.1 and 142.8 assigned to C-2 and C-3 respectively. The NMR data for compound
5.4 combined with its elemental composition indicated that it was most probably the previously reported physalin D.42 A comparison of its NMR data with the literature data of physalin D42 indicated that compound 5.4 was indeed physalin D (Table 5.1).
H O O O O
O HO H O O O OH OH 5.4
Compound 5.5 was also isolated as a white amorphous powder, and its HRFABMS
1 indicated a molecular formula of C28H31ClO10. Its H NMR spectrum revealed three singlets at δ
1.10 (s, 3H, H3-19), 1.15 (s, 3H, H3-28) and 1.76 (s, 3H, H3-21), a doublet of doublets at δ 2.47
(dd, 1H, J = 20.6, 5.1 Hα-4) and a broad doublet at δ 3.46 (br d, 1H, J = 20.6, Hβ-4). The
spectrum also exhibited a doublet of doublets at δ 2.88 (d, 1H, J =3.5 Hz, H-25), a doublet at δ
3.57 (d, 2H, J = 12.8 Hz, H2-27), a doublet of doublets at δ 4.24 (dd, 2H, J = 13.3, 4.6 Hz, H2-
27), a broad doublet at δ 3.87 (d, 1H, J = 3.6 Hz, H-6) corresponding to the methine proton of a
53 6β-equatorial hydroxyl group, and a doublet of doublet at δ 4.55 (dd, 2H, J = 3, 2 Hz, H-22). The spectrum also revealed the presence of one double bond with signals at δ 5.81 (dd, 1H, J = 10,
2.3 Hz, H-2) and δ 6.77 (ddd, 1H, J = 10, 4.8, 3.0 Hz, H-3). Like the spectrum of compound 5.4, the 13C NMR spectrum of compound 5.5 revealed the presence of two ketone peaks at δ 200.3
(C-1) and 209.8 (C-15), and two olefinic carbons at 127.1 and 143.0 assigned to C-2 and C-3 respectively. Its 13C NMR spectrum also showed the presence of seven oxygenated carbons at δ
60.5, 72.7, 82.4, 76.4, 78.7, 80.5 and 80.9 assigned to C-27, C-6, C-5, C-22, C-13, C-20, and C-
17 respectively. The main difference between the 13C NMR spectra of both compounds was the change in position of the ketone and C-5 peaks. The chemical shift for ketone C-1 was observed at δ 202.4 for Compound 5.4 and at δ 200.3 for compound 5.5. The chemical shift for C-5 was observed at δ 76.3 for compound 5.4 and at δ 82.4 for compound 5.5. The NMR data for compound 5.5 combined with its elemental composition and the difference in its NMR spectra compared to those of compound 5.4, and also by comparison of its NMR data with the literature data of physalin H45 (Table 5.1) indicated that it was indeed physalin H.
H O O O O
O HO H O O O Cl OH
Compound 5.6 was also isolated as a white amorphous powder and its HRFABMS
1 indicated a molecular formula of C30H34O12. Its H NMR spectrum revealed four three- proton
45 Makino, B.; Kawai, M. ; Ogura, T. ; Nakanishi, M. ; Yamamura, H. ; Butsugan; Structural revision of physalin H isolated from Physalis angulata, J. Nat. Prod. 1995, 58, 1668-1674
54 singlets at δ 1.15 (s, 3H, H3-19), 1.22 (s, 3H, H3-28), 1.71 (s, 3H, H3-21) and 1.81 (s, 3H,
CH3CO), a doublet of doublets at δ 2.93 (dd, 1H, J = 20.4, 7.8 H-2α), and a broad doublet at δ
3.41 (br d, 1 H, J = 20.6, H-2β). The spectrum also showed a doublet at δ 2.88 (d, 1H, J = 4.2 Hz,
H-25), a doublet at δ 3.57 (d, 2H, J = 13.3 Hz, H2-27), a doublet of doublets at δ 4.24 (dd, 2H, J
= 13.3, 4.6 Hz, H2-27), a broad doublet at δ 4.4 ( br d, 1 H, J = 3.0 Hz, H-6) and doublet at δ 5.19
(d, 1H, J = 3.9 Hz, H-6), a doublet of doublets at δ 5.76 (dd, 1H, J = 10, 2.3 Hz, H-4) and δ 6.70
(ddd, 1H, J = 10, 4.8, 3.0 Hz, H-3). Its 13C NMR spectrum revealed the presence of two ketone peaks at δ 203.0 (C-1) and 209.7 (C-15), and two olefinic carbons at 127.1 and 142.5 assigned to
C-2 and C-3 respectively. This spectrum also showed the presence of seven oxygenated carbons at δ 60.5, 65.1, 66.5, 76.3, 78.7, 80.5 and 80.8 assigned to C-27, C-6, C-5, C-22, C-13, C-20, and
C-17 respectively, a ketal at δ 106.5, two lactone carbonyl peaks at δ167.4 and 171.4, and an ester carbonyl at δ 168.8. The NMR data for compound 5.6 combined with its elemental composition, and a comparison of its 1H NMR data with the literature data of physalin D-6- acetate46 indicated that compound 5.6 was indeed physalin D-6-acetate. There is no published
13C NMR data for this compound.
H O O O O
O HO H O O O OH 5.6 OAc
46 Row, L. R. ; Reddy, K. S.; Sarma, S. ; Matsuura, T.; Nakaashima, R.; New physalins from Physalis angulata and Physalis lancifolia. Structure and reactions of phasylins D, I, G and K; Phytochem. 1980, 19, 1175-1181
55 Compound 5.7 was also isolated as a white amorphous powder and its HRFABMS
1 indicated a molecular formula of C30H33ClO11. Like that of compound 5.6, its H NMR spectrum
revealed four three - proton singlets at δ 1.16 (s, 3H, H3-19), 1.17 (s, 3H, H3-28), 1.81 (s, 3H, H3-
21) and 1.81 (s, 3H, CH3CO) assigned to an acetoxyl group. The doublet of doublets at δ 2.47
and a broad doublet at δ 3.46 were assigned to a methylene at C-2. Its 1H NMR spectrum also
had a doublet of doublets at δ 3.31 (dd, 1H, J = 15.5, 5 Hz, H-4α), a doublet of doublets of
doublets δ 2.22 (ddd, 1H, J = 20, 11.5, 2.5, Hβ-4), a broad doublet at δ 4.54 ( br d, 1 H, J = 3.0
Hz, H-6), and a doublet at δ 5.36 (d, 1H, J = 4.9 Hz, H-6). Its 13C NMR spectrum showed the presence of seven oxygenated carbons at δ 60.5, 65.2, 89.6, 76.3, 78.7, 80.5 and 80.8 assigned to
C-27, C-6, C-5, C-22, C-13, C-20, and C-17 respectively, a ketal at δ 106.5, two lactone carbonyl peaks at δ 167.3 and 171.7, and an ester carbonyl at δ 169.3. The main difference between the
13C NMR spectra of compounds 5.6 and 5.7 was the change in the chemical shift for the C-5 peak. In compound 5.6, C-5 signal was at δ 66.5, but in compound 5.7 it was shifted to δ 89.5.
The NMR data for compound 5.7 combined with its elemental composition indicated that it was the previously reported physalin H-6 acetate.46
H O O O O
O HO H O O O Cl OAc 5.7
56 Table 5.1 13C NMR spectra of compounds 5.1-5.4
Carbon 5.1 Lit.Data42 5.2 Lit.Data43 5.3 5.4 Lit.Data42 1 202.4 202.6 209.2 208.2 204.8 204.3 204.3 2 126.9 127.1 39.3 39.6 125.8 127.1 127.1 3 146.2 146.4 122.5 121.3 140.0 142.8 142.8 4 32.3 32.5 125.8 126.6 116.5 35.1 35.1 5 135.6 135.8 140.4 139.7 157.9 76.3 76.3 6 123.4 123.6 128.1 128.9 71.6 72.5 72.5 7 24.4 24.6 24.6 25.9 24.4 26.6 26.6 8 39.9 40.3 39.5 39.2 40.5 38.2 38.2 9 33.1 33.3 31.5 33.1 34.2 29.8 29.8 10 51.9 52.2 54.2 56.5 53.5 53.4 53.4 11 24.4 24.6 24.8 24.7 24.4 24.7 24.7 12 25.6 25.7 25.4 26.6 25.2 25.7 25.7 13 78.2 78.4 78.3 79.8 78.1 78.5 78.5 14 106.3 106.5 106.4 107.7 106.4 106.8 106.8 15 209.4 209.6 209.9 214.1 209.5 209.8 209.8 16 54.1 54.4 54.2 55.1 53.7 53.8 53.8 17 80.7 80.9 80.7 80.4 80.7 80.4 80.4 18 171.8 172.0 171.7 172.2 171.7 171.8 171.8 19 17.8 17.0 18.4 25.4 18.3 13.2 13.2 20 80.3 80.5 80.3 81.1 80.4 80.6 80.6 21 21.7 21.9 21.7 21.5 21.6 21.6 21.6 22 76.3 76.5 76.3 77.0 76.3 76.3 76.3 23 31.4 31.6 31.6 32.1 31.2 31.2 31.2 24 30.5 30.7 30.5 31.2 30.4 30.4 30.4 25 49.4 49.6 49.3 50.6 49.3 49.3 49.3 26 167.3 167.5 167.2 166.7 167.2 167.3 167.3 27 60.6 60.8 60.7 60.7 60.7 60.4 60.4 28 24.4 24.6 24.4 19.3 18.2 24.4 24.4
57 Table 5.2 13C NMR spectra of compounds 5.5-5.7
Carbon 5.5 Lit.Data45 5.6 5.7 1 200.3 200.3 203.2 203.0 2 127.2 127.2 127.1 127.1 3 142.9 142.9 144.9 142.6 4 36.9 36.9 31.3 31.3 5 82.4 82.3 64.5 89.6 6 72.7 72.6 65.9 65.3 7 26.8 26.7 24.4 24.4 8 38.4 38.4 37.9 37.8 9 30.9 30.9 30.5 30.5 10 53.8 53.8 53.6 53.6 11 24.3 24.3 24.4 24.4 12 25.8 25.8 26.0 25.8 13 78.4 78.4 78.7 78.7 14 106.4 106.4 106.5 106.6 15 209.8 209.6 209.8 209.7 16 53.8 53.9 53.8 54.0 17 80.9 80.8 81.0 80.8 18 171.7 171.5 171.5 171.7 19 14.1 14.0 13.0 13.2 20 80.5 80.4 80.6 80.5 21 21.7 21.6 21.5 21.6 22 76.3 76.3 76.3 76.3 23 31.3 31.3 30.5 30.5 24 30.5 30.4 29.7 30.0 25 49.3 49.4 49.3 49.3 26 167.3 167.3 167.4 167.3 27 60.6 60.5 60.6 60.6 28 24.4 24.4 24.4 24.4
6-COCH3 168.6 169.3
6-COCH3 24.1 21.2
58 5-3 Experimental Section
5.3.1 General Experimental Procedures
The NMR spectra were obtained on either a JEOL Eclipse 500 spectrometer or on a
Varian Inova 400 spectrometer. The mass spectra were obtained on a JEOL JMS-HX-110 instrument. A flash chromatograph from Biotage Inc. was used for flash chromatography. HPLC was performed on a Shimadzu LC-10AT instrument using a Varian Dynamax C18 column (250
´ 10 mm). C-18 SPE columns were obtained from Supelco.
5.3.2 Plant Material
The extracts, root, stems, twigs fruits and leaves of Physalis angulata L. (Solanaceae) were collected by Iwan Derveld from the rainforest of Botopasi on the Suriname River in
Suriname, in the vicinity of Vohimena on February 3rd, 1998. The roots, stem, twigs, leaves and fruits of the dried plant material were extracted with EtOAc and then with MeOH to yield extracts 980288E and 980288M.
5. 3. 3 Extraction and Isolation
Extracts 980288E and 980288M from Physalis angulata L. (Solanaceae) (2 g) were partitioned between hexane and 60% aqueous MeOH, and the latter extract was diluted to 50% aqueous MeOH and extracted with CH2Cl2. All the resulting fractions were evaporated to dryness and tested for their biological activity against Plasmodium falciparum. Only CH2Cl2 fraction was active with an IC50 value of 9 µg/mL. Thin layer chromatography (TLC) revealed that both the CH2Cl2 fractions of extract 980288E and 980288M contained almost the same
59 constituents and had the same IC50 value. Therefore they were combined and purified through
normal phase preparative HPLC using CH2Cl2/CH3CN gradient (3% to 100% CH3CN) as mobile phase to yield compounds 5.1-5.7.
The pure compounds 5.1-5.7 were tested in two different antimalarial assays. In the assay conducted at the “Instituto de Medicina Tropical y Ciencias de la Salud” in Panama, all the compounds were recorded as active; but without any IC50 values. In a later assay by our collaborator in Madagascar, the compounds were reported as having no activity. It is believed that this result may have been due to problems with the assay in Madagascar, but unfortunately the lack of samples has presented a detailed reinvestigation of this question.
Compound 5.1: white amorphous powder(2 mg); 1H NMR (500 MHz, DMSO) δ 5.80
(dd, 1H, J = 10, 2.3 Hz, H-2), 6.89 (ddd, 1H, J = J = 10, 4.8, 2.2 Hz, H-3), 2.89 (dd, 1H, J = 20,
5 Hz, H-4α), 3.27 (br d, 1H, J = 20 Hz, H-4β), 5.58 (br d, 1H, J = 6.2 Hz, H-6), 1.97 (m, 1H, H-
7α), 2.21 (m, 1H, H-7β), 1.92 (m, 1H, H-8), 2.95 (dd, 1H, J = 11.6, 9 Hz, H-9), 2.18 (m, 1H, H-
11α), 0.96 (m, 1H, H-11β), 2.17 (m, 1H, H-12α), 1.44 (br dd, 1H, J = 16, 9 Hz, H-12β), 6.30 (s,
1H, OH [C-13]), 2.86 (s, 1H, H-16), 1.15 (s, 1H, Me-19), 1.78 (s, 1H, Me-21), 4.57 (dd, 2H, J =
3, 2 Hz, H-22), 1.96 (dd, 1H, J = 15, 2 Hz, H-23), 2.14 (dd, 2H, J = 15, 2 Hz, H-23), 2.88 (br d,
1H, J = 4.5, 1 Hz, H-25), 3.60 (br d, 1H, J = 12.8 Hz, H-27α), 4.26 (dd, 1H, J = 13.5, 4 Hz, H-
27), 1.10 (s, Me-28). 13C NMR (Table 5.1). HRFABMS found m/z 511.1965 [M+H]+; calcd for
C28H31O9, 511.1968.
Compound 5.2: white amorphous powder(3 mg); 1H NMR (500 MHz, DMSO) δ 2.61
(dd, 1H, J = 20.0, 5.0 Hz, H-2α), 3.40 (br d, 1H, J = 10, 4.8, 2.2 Hz, H-2β), 5.63 (m, 1H, H-3),
60 6.05 (dd, 1H, J = 10.0, 2.0 Hz, H-4), 5.67 (br d, 1H, J = 3.0 Hz, H-6), 2.30 (m, 1H, H-7α), 2.35
(m, 1H, H-7β), 1.92 (m, 1H, H-8), 3.0 (t, 1H, J = 9.6 Hz, H-9), 2.18 (m, 1H, H-11α), 1.0 (m, 1H,
H-11β), 2.17 (m, 1H, H-12α), 1.44 (br dd, 1H, J = 16, 9 Hz, H-12β), 6.30 (s, 1H, OH [C-13]),
2.85 (s, 1H, H-16), 1.15 (s, 1H, Me-19), 1.78 (s, 1H, Me-21), 4.57 (dd, 2H, J = 3, 2 Hz, H-22),
1.92 (dd, 1H, J = 15, 2 Hz, H-23), 2.10 (dd, 2H, J = 15, 2 Hz, H-23), 2.88 (br d, 1H, J = 4.5, 1
Hz, H-25), 3.60 (br d, 1H, J = 12.8 Hz, H-27α), 4.26 (dd, 1H, J = 13.5, 4 Hz, H-27), 1.10 (s, Me-
13 + 28). C NMR (Table 5.1). HRFABMS found m/z 511.1964 [M+H] ; calcd for C28H31O9,
511.1968.
Compound 5.3: white amorphous powder (5 mg); 1H NMR (500 MHz, DMSO) δ 5.93
(dd, 1H, J = 9.5Hz, H-2), 7.03 (dd, 1H, J = 11, 7 Hz, H-3), 6.17 (d, 1H, J = 6.5 Hz, H-4α), 4.48
(dd, 1H, J = 3, 2 Hz, H-6), 5.65 (d, 1H, J = 5 Hz, OH [C-6]), 1.95 (m, 1H, H-7α), 2.04 (m, 1H,
H-7β), 2.27 (dt, 1H, J = 11.4, 11.5, 4 Hz, H-8), 3.34 (dd, 1H, J = 11.6, 8 Hz, H-9), 2.13 (m, 1H,
H-11α), 0.96 (m, 1H, H-11β), 1.89 (m, 1H, H-12α), 1.44 (br dd, 1H, J = 16, 9 Hz, H-12β), 6.05
(s, 1H, OH [C-13]), 2.82 (s, 1H, H-16), 1.15 (s, 1H, Me-19), 1.76 (s, 1H, Me-21), 4.55 (dd, 2H, J
= 3, 2 Hz, H-22), 1.92 (dd, 1H, J = 15, 2 Hz, H-23), 2.10 (dd, 2H, J = 15, 2 Hz, H-23), 2.88 (d,
1H, J = 4.5, 1 Hz, H-25), 3.57 (br d, 1H, J = 12.8 Hz, H-27α), 4.24 (dd, 1H, J = 13.5, 4 Hz, H-
27), 1.10 (s, Me-28). 13C NMR (Table 5.1). HRFABMS found m/z 527.1917 [M+H]+; calcd for
C28H31O10, 527.1917.
Compound 5.4: white amorphous powder (9 mg); 1H NMR (500 MHz, DMSO) δ 5.70
(dd, 1H, J = 10, 2.3 Hz, H-2), 6.62 (ddd, 1H, J = 10, 4.8, 2.0 Hz, H-3), 1.98 (dd, 1H, J = 20.6,
5.1 Hz, H-4α), 3.11 (brd, 1H, J = 20.6 Hz, H-4β), 4.25 (s, 1H, OH [C-5]), 3.49 (br d, 1H, J = 3
61 Hz, H-6), 4.90 (d, 1H, J = 4 Hz, OH [C-6]), 1.80 (m, 1H, H-7α), 1.84 (m, 1H, H-7β), 2.20 (dt, 1H,
J = 11.4, 11.5, 4 Hz, H-8), 3.11 (dd, 1H, J = 11.6, 8 Hz, H-9), 2.11 (m, 1H, H-11α), 0.94 (m, 1H,
H-11β), 2.10 (m, 1H, H-12α), 1.44 (br dd, 1H, J = 16, 9 Hz, H-12β), 5.76 (s, 1H, OH [C-13]),
2.77 (s, 1H, H-16), 1.10 (s, 1H, Me-19), 1.76 (s, 1H, Me-21), 4.56 (dd, 2H, J = 3, 2 Hz, H-22),
1.92 (dd, 1H, J = 15, 2 Hz, H-23), 2.10 (dd, 2H, J = 15, 2 Hz, H-23), 2.88 (d, 1H, J = 4.5, 1 Hz,
H-25), 3.57 (br d, 1H, J = 12.8 Hz, H-27α), 4.24 (dd, 1H, J = 13.5, 4 Hz, H-27), 1.10 (s, Me-28).
13 + C NMR ( Table 5.1). HRFABMS found m/z 545.2019 [M+H] ; calcd for C28H33O11, 545.2022.
Compound 5.5: white amorphous powder (3 mg); 1H NMR (500 MHz, DMSO) δ 5.81
(dd, 1H, J = 10, 2.3 Hz, H-2), 6.77 (ddd, 1H, J = 10, 4.8, 2.0 Hz, H-3), 2.47 (dd, 1H, J = 20.6,
5.1 Hz, H-4α), 3.46 (brd, 1H, J = 20.6 Hz, H-4β), 3.87 (br d, 1H, J = 3.6 Hz, H-6), 5.65 (d, 1H, J
= 5 Hz, OH [C-6]), 1.95 (m, 1H, H-7α), 2.04 (m, 1H, H-7β), 2.27 (dt, 1H, J = 11.4, 11.5, 4 Hz,
H-8), 3.34 (dd, 1H, J = 11.6, 8 Hz, H-9), 2.13 (m, 1H, H-11α), 0.96 (m, 1H, H-11β), 1.89 (m, 1H,
H-12α), 1.44 (br dd, 1H, J = 16, 9 Hz, H-12β), 6.05 (s, 1H, OH [C-13]), 2.82 (s, 1H, H-16), 1.15
(s, 1H, Me-19), 1.76 (s, 1H, Me-21), 4.55 (dd, 2H, J = 3, 2 Hz, H-22), 1.92 (dd, 1H, J = 15, 2 Hz,
H-23), 2.10 (dd, 2H, J = 15, 2 Hz, H-23), 2.88 (d, 1H, J = 4.5, 1 Hz, H-25), 3.57 (br d, 1H, J =
12.8 Hz, H-27α), 4.24 (dd, 1H, J = 13.5, 4 Hz, H-27), 1.10 (s, Me-28). 13C NMR (Table 5.2)
+ HRFABMS found m/z 562.1681[M+H] ; calcd for C28H32ClO10, 563.1684.
Compound 5.6: white amorphous powder (2.5 mg); 1H NMR (500 MHz, DMSO) δ 5.81
(dd, 1H, J = 10, 2.3 Hz, H-2), 6.77 (ddd, 1H, J = 10, 4.8, 2.0 Hz, H-3), 2.47 (dd, 1H, J = 20.6,
5.1 Hz, H-4α), 3.46 (brd, 1H, J = 20.6 Hz, H-4β), 3.87 (br d, 1H, J = 3.6 Hz, H-6), 1. [C-6]), 1.95
(m, 1H, H-7α), 2.04 (m, 1H, H-7β), 2.27 (dt, 1H, J = 11.4, 11.5, 4 Hz, H-8), 3.34 (dd, 1H, J =
62 11.6, 8 Hz, H-9), 2.13 (m, 1H, H-11α), 0.96 (m, 1H, H-11β), 1.89 (m, 1H, H-12α), 1.44 (br dd,
1H, J = 16, 9 Hz, H-12β), 6.05 (s, 1H, OH [C-13]), 2.82 (s, 1H, H-16), 1.15 (s, 1H, Me-19), 1.76
(s, 1H, Me-21), 4.55 (dd, 2H, J = 3, 2 Hz, H-22), 1.92 (dd, 1H, J = 15, 2 Hz, H-23), 2.10 (dd, 2H,
J = 15, 2 Hz, H-23), 2.88 (d, 1H, J = 4.5, 1 Hz, H-25), 3.57 (br d, 1H, J = 12.8 Hz, H-27α), 4.24
(dd, 1H, J = 13.5, 4 Hz, H-27), 1.10 (s, Me-28). 13C NMR (Table 5.2). HRFABMS found
+ 604.1708 m/z [M+H] ; calcd for C30H33ClO11, 604.1711.
Compound 5.7: white amorphous powder (1.9 mg). 1H NMR (500 MHz, DMSO) δ 5.75
(dd, 1H, J = 10, 2.3 Hz, H-2), 6.70 (ddd, 1H, J = 10, 4.8, 2.0 Hz, H-3), 2.47 (dd, 1H, J = 20.6,
5.1 Hz, H-4α), 3.46 (brd, 1H, J = 20.6 Hz, H-4β), 3.87 (br d, 1H, J = 3.6 Hz, H-6), 5.65 (d, 1H, J
= 5 Hz, OH [C-6]), 1.95 (m, 1H, H-7α), 2.04 (m, 1H, H-7β), 2.27 (dt, 1H, J = 11.4, 11.5, 4 Hz,
H-8), 3.34 (dd, 1H, J = 11.6, 8 Hz, H-9), 2.13 (m, 1H, H-11α), 0.96 (m, 1H, H-11β), 1.89 (m, 1H,
H-12α), 1.44 (br dd, 1H, J = 16, 9 Hz, H-12β), 6.05 (s, 1H, OH [C-13]), 2.82 (s, 1H, H-16), 1.15
(s, 1H, Me-19), 1.71 (s, 1H, Me-21), 4.55 (dd, 2H, J = 3, 2 Hz, H-22), 1.92 (dd, 1H, J = 15, 2 Hz,
H-23), 2.10 (dd, 2H, J = 15, 2 Hz, H-23), 2.88 (d, 1H, J = 4.5, 1 Hz, H-25), 3.57 (br d, 1H, J =
12.8 Hz, H-27α), 4.24 (dd, 1H, J = 13.5, 4 Hz, H-27), 1.10 (s, Me-28). 13C NMR (Table 5.2).
+ HRFABMS found m/z 586.2125 [M+H] ; calcd for C30H35O12, 587.2128.
63 Physalis angulata L
E/M 980288 Potential antimalarial
2 g IC50 = 11 µg/mL
partition hexane/60% aqueous MeOH
partition CH2Cl2/50% aqueous MEOH Hexane Yield 606.7 mg
% 30.3 MeOH CH2Cl2 216 mg IC50 = Not active Yield 1094 mg % 54.7 10.8
Not active IC50 = 9 µg/mL
C18 flash chr. MeOH/H2O: 8/2
370 mg 680 mg
Active (no IC50 available)Not active
150 mg used Preparative HPLC, followed by anatical HPLC CH2Cl2/CH3CN gradient
5.1 5.25.35.4 5.5 5.65.7 Yield 2 mg3mg5 mg9 mg3 mg2.5 mg1.9 mg
Note: All compounds isolated were around 95% pure. The low yields of isolated compounds are due to solubility problems. The samples were dissolved in DMSO before injection
Figure 5.1: Bioassay guided fractionation of Physalis angulata L
64 VI. CARDENOLIDE GLYCOSIDES FROM ROUPELLINA BOIVINII
6.2 Introduction
Roupellina boivinii (Baill) Pichon belongs to the Apocynaceae family. Previous chemical
studies of this plant had revealed the presence of cardenolide glycosides. An extract of
Roupellina boivinii was received from the National Cancer Institute (NCI) through the ICBG
program in order to investigate its potential anticancer activity. The crude extract had an IC50 of
3 µg/mL against the A2780 ovarian cancer cell line.
6.2 Results and Discussion
The extract of Roupellina boivinii was subjected to solvent partitioning between hexane
and aqueous MeOH, and the aqueous MeOH fraction was then extracted with CH2Cl2. All the
phases were evaporated under reduced pressure and the resulting products tested for their
biological activities against the A2780 ovarian cancer cell line. The CH2Cl2 and MeOH fractions
were both active fractions. The MeOH fraction was then partitioned between BuOH and water,
and both fractions tested for their activity. The BuOH fraction was the only active fraction. Both
the CH2Cl2 and BuOH fractions were passed through short C18 chromatography columns and
then purified by reversed phase preparative HPLC using MeOH/H2O (6/4) as mobile phase to yield the three known cardenolide glycosides (6.1, 6.2 and 6.4) and three new cardenolide glycosides 6.3, 6.5 and 6.6.
Compound 6.1 was isolated as an amorphous substance, and HRFABMS indicated a
1 molecular formula of C29H42O8. Its H NMR spectrum revealed one singlet at δ 0.81 (s, 3H), δ
2.81 (dd, 1H, J = 9.1, 5.2 Hz), δ 4.91 (dd, 1H, J = 7.8, 2.0 Hz), δ 5.01 (dd, 1H, J = 18.5, 1.8 Hz)
and δ 5.88 (brs, 1H) and also a singlet at δ 9.98 corresponding to an aldehyde. These peaks,
65 coupled with the one singlet (1 x CH3), suggested that compound 6.1 could be a cardenolide since these chemical shifts are characteristic of the chemical shifts of the aglycon portion of cardenolides. The doublet of doublets at δ 2.81 indicated that the stereochemistry of the lactone at C-17 was β.47 The 13C NMR spectrum of compound 6.1 showed three peaks at δ 117.9, 177.19 and δ 178.20; these signals are indicative of an α,β-unsaturated γ-lactone, and are always present
in the 13C NMR spectra of cardenolides. The 1H NMR spectrum had signals for one anomeric proton at δ 4.86 (dd, J = 8.7, 3.2 Hz, 1′-H) indicating the presence of one sugar. This conclusion was confirmed by its 13C NMR spectrum, which contained one peak for an anomeric carbon at δ
97.54. The presence of a doublet at δ 1.21 (d, 3H, J = 6.6 Hz, 6′-H) indicated that the sugar was a
6-deoxy-sugar. Its 13C NMR spectrum showed signals for seven oxygenated carbons at δ 70.2,
70.4, 71.1, 75.3, 77.4, 85.8 and 97.5 and included three signals from the aglycone moiety, meaning that the sugar had four oxygenated carbons. The structure of the sugar was identified by
ROESY correlations (Figure 6.1) and a 1D-TOCSY spectrum, and comparison of proton-proton coupling constants with those of β-D-boivinopyranoside.48 The structure of the sugar was then confirmed by comparing its 13C chemical shifts with those of β-D-boivinopyranoside. 48 The
carbon data of the aglycone were assigned using HMQC and HMBC (see spectrum in Appendix)
correlations depicted in Figure 6.1. The aldehyde peak at δ 9.98 assigned to H-19 correlated with
carbon peaks at δ 52.8, 44.1, 37.0 and 49.6 assigned to C-10, C-5, C-1 and C-9 respectively. The
proton peak at δ 3.71 (m) assigned to H-3 correlated with carbon peaks at δ 97.54 corresponding
to the anomeric carbon, 29.9, 32.1 assigned to C-2 and C-4, and also correlated to C-5. The
singlet at δ 0.81 (H-18) showed correlations with carbon peaks at δ 85.8 (C-14), 50.7 (C-13),
47 End, H.; Warashina, T.; Noro, T.; Castro, V. H.; Mora, G. A.; Poveda, L. J.; Sanchez, P. E. Cardenolide glycosides from Thevetia ahouai (LINN.) A.D.C. Chem. Pharm. Bull. 1997, 45, 1536-1538 48 Nakamura, T.; Goda, Y.; Sakai, S.; Kondo, K.; Akiyama, H.; and Toyoda, M. ; Cardenolide glycosides from seeds of Corchorus olitorius. Phytochemistry 1998, 49, 2097-2101
66 40.4 (C-12) and 51.9 (C-17). The proton peak at δ 2.81 (H-17) showed correlations with carbon peaks at δ 178.2 (C-20), 75.3 (C-21) and 117.9 (C-22). Based on these HMBC correlations, the aglycone portion of compound 6.1 was determined to be corotoxigenin, and its carbon data were compared to those of uzarigenin. The difference between corotoxigenin and uzarigenin is that corotoxigenin has an aldehyde at C-19 and uzarigenin has a methyl group. The NMR data for compound 6.1 combined with its elemental composition indicated that it was 5-α- corotoxigenin
–β-D-boivinoside. A literature search confirmed that compound 6.1 had been isolated from the
49 same plant in 1961, but no NMR data were reported at that time. It had an IC50 of 0.08 µg/mL
against the A2780 cell line.
O O
22 O O 18 21 12 O 17 19 13 O 1 9 14 OH 6' 10 OH H C 3 H OH H OH 4' 3 5' O O O H O H 6 3' 2' H H 1' OH 6.1 OH H HMBC correlations
O
O
O
OH H OH O H O H H H OH H ROESY correlations
Figure 6.1 Structure ROESY and HMBC correlations compound 6.1.
49 Russel, J. H.; Schindler, O.; Reichstein, T. Die cardenolide der blättter von Roupellina boivinii.2. Helv. Chim. Acta, 1961, 44, 1315-30
67 Compound 6.2 was also isolated as an amorphous substance, and its HRFABMS
indicated a molecular formula of C30H44O8. Its NMR spectrum showed a singlet peak at δ 0.97, a
triplet at δ 3.16 (t, 1H, J = 9.6 Hz, H-17), a doublet of doublets at δ 4.94 (dd, 1H, J = 19, 1.3 Hz,
H-21), and also a doublet of doublet at δ 4.84 (dd, 1H, J = 19, 1.2 Hz, H-21) and δ 5.93 (brs, 1H,
H-22). These peaks, coupled with the presence of one singlet (CH3), suggested that compound
6.2 could be a cardenolide, since these chemical shifts are characteristic of the aglycon portion of cardenolides. The triplet at δ 3.17 indicated that the lactone stereochemistry at C-17 was
α.orientation as previously reported.50,51 It 1H NMR had a single peak at d 9.98, indicating the presence an aldehyde on the aglycone of compound 6.2. The position of aldehyde is always reported at C-19. The proton spectrum revealed the presence of only one anomeric proton at δ
4.78 (dd, J = 8.7, 3.2 Hz, 1′-H) and a singlet at δ 3.36 (s, 3H) representing one methoxy group.
Its 13C NMR spectrum confirmed the presence of only one sugar moiety with a single anomeric carbon peak at δ 97.63 and the aldehyde at C-19 with a peak at δ 210.35. The presence of a doublet at δ 1.20 (d, 3H, J = 6.6 Hz, 6′-H) indicated that the sugar was also a 6-deoxy-sugar. Its
13C NMR spectrum showed seven oxygenated carbons at δ 68.2, 70.6, 75.6, 77.4, 80.3, 86.5 and
97.6 and a methoxy peak at δ 57.2; these resonances included three from the aglycone moiety.
The stereochemistries of the sugars were determined using ROESY and the correlations depicted in Figure 6.1. The carbon data of the aglycone were also assigned using HMQC and HMBC correlations depicted in Figure 6.2. The HMBC correlations described in compound 6.1 were also observed. The major difference in the aglycone of the two compounds was the chemical shift at δ 2.81 (H-17), indicating that the aglycone of compound 6.1 was 17β corotoxigenin, and
50 Kawaguchi, K.; Hirotani, M.; Fuuraya, T. Biotransformation of ditgitoxigenin by cell suspension cultures of Strophantus divaricatus. Phytochemistry, 1991, 30, 1503 51Yamauchi, T.; Abe, F.; Wan, A. S.C. Cardenolide monoglycosides from the leaves of Cerbera odollam and Cebera manghas (Cerbera. III). Chem. Pharm. Bull. 1987, 35, 2744-249
68 the chemical shift at δ 3.17 (H-17), indicating that the aglycone of the compound 6.2 was 17α
corotxigenin. The carbon data of compound 6.2 were assigned based on HMBC correlations
shown in Figure 6.2 (see the full spectrum in the appendix). The chemical shift at δ 3.17 (H-17)
showed correlations with carbon peak at δ 30.6 (C-12), δ 49.9 (C-13), 86.5 (C-14), 32.1 (C-15)
and 25.2 (C-16). The NMR data for compound 6.2 combined with its elemental composition indicated that it was 17α-corotoxigenin-β-D-sarmentoside, also known as madagascoside, isolated from the same plant in 1961,49 although no NMR data were reported at that time. It had an IC50 of 2.0 µg/mL against the A2780 cell line.
O O
22 O O 18 21 12 O 17 19 13 O 1 9 14 OH 6' 10 OH H C 3 H OH H OH 4' 3 5' O O O H 6 O 3' 2' H H H 1' OCH3 6.2 OCH3 H HMBC correlations
O
O
O
OH H OH O H O H H H OCH3 H ROESY correlations
Figure 6.2 Structure, ROESY and HMBC correlations of compound 6.2.
69 Compound 6.3 was also isolated as an amorphous substance, and its HRFABMS
1 indicated a molecular formula of C36H56O12. Its H NMR spectrum also showed two singlets at δ
0.82 (s, 3H, H3-18), and 1.03 (s, 3H, H3-19), and a triplet at δ 3.17 (t, 1H, J = 9.6 Hz, H-17), δ
4.84 (dd, 1H, J = 18.1, 1.8 Hz, H-21), δ 4.94 (dd, 1H, J = 19.7, 1.2 Hz, H-21) and δ 5.93 (brs, 1H,
H-22). These peaks taken together suggested that compound 6.3 was also a cardenolide. The triplet at δ 3.17 also indicated that the lactone stereochemistry at C-17 was in the α- orientation.50,51 Its 1H NMR spectrum also revealed the presence of two anomeric protons at δ
4.28 (d, 1H, J= 7.8 Hz 1-H), and 4.82 (dd, 1H, J = 8.7, 3.2 Hz, 1′-H). A doublet at δ 1.24 (d, 3H,
J = 6.6 Hz, 6′-H) indicated that one sugar was a 6-deoxy-sugar, and a methoxy peak at δ 3.38 was observed. The presence of two sugars was confirmed by its 13C NMR spectrum, which showed resonances for two anomeric carbons at δ 97.61 (C-1′) and 102.18 (C-1″). Its 13C NMR spectrum showed signals for ten oxygenated carbons at δ 63.0, 70.34, 71.9, 74.9, 75.6, 76.5, 77.9,
78.1, 78.4, 87.02, 97.6 and 102.2 including three signals from the aglycone moiety. The structure and the connectivity of the two sugars were confirmed by the COSY and HMBC correlations depicted in Figure 6.3. The stereochemistry of the sugars was determined using ROESY and 1D-
TOCSY and by measuring proton-proton coupling constants. The structures of the sugars were confirmed by comparison of their carbon chemical shifts to those of the sugars of oleandrigenin
β-D-glucosyl-β-D-sarmentoside. 52 The carbon data of the aglycone of compound 6.3 were assigned based on the HMBC correlations depicted in Figure 6.3 and confirmed by comparison of these carbon assignments to those of 5α,17α-uzarigenin previously reported.53 Both data were close enough to conclude that the aglycone of compound 6.3 was indeed 5α,17α-uzarigenin. The
52 Abe, F.; Yamauchi, T. Cardenolide trioside of oleander leaves. Phytochemistry, 1992, 31, 2459-2463 53 Yamauchi, T.; Abe, F.; Nishi, M. Carbon -13 NMR of 5α-cardenolide. Chem. Pharm. Bull. 1978, 26, 2894-2896
70 NMR data for compound 6.3 combined with its elemental composition indicated that it was the new cardenolide glycoside, 5α,17α-uzarigenin-3-O-[β-D-glucopyranosyl-(1→4)-β-D
-sarmentoside]. It had an IC50 of 2.0 µg/mL against the A2780 cell line.
O
O
OH OH O O HO H3C H OH OH O O H 6.3 OCH3 O
O
OH OH O O HO H3C H OH OH O O H
OCH3 HMBC correlations O
O
OH OH O H O HO H3C OH OH O H O
OCH3 H ROESY correlations
Figure 6.3 Structure, ROESY and HMBC correlations of compound 6.3
71 Compound 6.4 was also isolated as an amorphous white powder. Its HRFABMS
1 indicated a molecular formula of C30H46O8. Its H NMR spectrum revealed two singlets at δ 0.83
(s 3H, H3-18) and δ 1.00 (s, 3H, H3-19), and a doublet of doublets at δ 2.80 (dd, 1H, J = 5.1, 8.6
Hz, H-17) which indicated that compound 6.4 had the same 17β aglycone as compound 6.1. Its
1H NMR spectrum also showed one anomeric proton at δ 5.38 (brd, 1H, J = 1.2Hz, H-1′). The presence of a single sugar was confirmed by its 13C NMR spectrum, which showed a single signal for an anomeric carbon at δ 99.85. Its 13C NMR spectrum also showed signals for eight oxygenated carbons at δ 70.3, 73.1, 73.5, 73.7, 77.3, 77.7, 84.3, and 99.9 including three from the aglycone moiety. The stereochemistry of the sugars was determined using ROESY and 1D-
TOCSY and by comparison of proton-proton coupling constants and the carbon data to those of
α-L-rhamnoside.54 The carbon data of the aglycone of compound 6.4 were assigned based on
HMBC correlations and confirmed by comparison these carbon assignments to those of 5α,17β uzarigenin previously reported.55 Both data were close enough to conclude that the aglycone of compound 6.3 was indeed 5α,17β-uzarigenin. The NMR data for compound 6.4, combined with its elemental composition, indicated that it was uzarigenin 3-O-α-L-rhamnoside previously
56 reported. It had an IC50 of 0.08 µg/mL against the A2780 cell line.
54 Hyun, J. W.; Shin, J. E.; Lin, K. H.; Sung, M. S.; Park, J. W.; Yu, J. H.; Kim, B. K.; Paik, W. H.; Kang, S. S.; Park, G. J. Evomonoside: The cytotoxic cardiac glycoside from Lepidium apetalum, Planta Med. 1995, 61, 294-295 55 Hanada, R.; Abe, F.; Yamauchi, T. Steroid glycoside from the roots of Nerium odorum. Phytochemistry, 1992, 31,3183-3187 56 Cheung, H. T. A.; Brown, L. Boutagy, J.; Thomas, R.; Cardenolide analogues. Part 12. 13C NMR of semi- synthetic glycosides and side-chain modified genins. J. Chem. Sc. Perkin I, 1981, 1773-1778
72 O
O
H
H OH O H O 6.4
HO HO OH
Compound 6.5 was isolated as a white amorphous powder, and HRFABMS indicated a
1 molecular formula of C35H52O13. Its H NMR spectrum revealed one singlet at δ 0.81 (s, 3H, H3-
18), δ 2.81 (dd, 1H, J = 9.1, 5.2 Hz, H-17β), δ 4.91 (dd, 1H, J = 7.8, 2.0 Hz, H-21), δ 5.01 (dd,
1H, J = 18.5, 1.8 Hz, H-21) and δ 5.88 (brs, 1H, H-22) and also a singlet at δ 9.98 corresponding
to aldehyde at C-19. The doublet at δ 2.81 indicated that the stereochemistry at C-17 was β.
These peaks, coupled with one methyl singlet, indicated that compound 6.5 had the same
aglycon portion as compound 6.1. The 1H NMR spectrum had signals for two anomeric protons at δ 4.28 (d, J = 7.8 Hz, H-1″) and δ 4.86 (dd, J = 8.7, 3.2 Hz, 1′-H). The 13C NMR spectrum of compound 6.5 showed peaks at δ 177.19 for C-23 and at δ 178.20 for C-20 and at δ 210.4 for the aldehyde at C-19. Its 13C NMR spectrum confirmed the presence of two anomeric carbons at δ
97.60 (C-1′) and δ 102.21 (C-1″). The presence of a doublet at δ 1.25 (d, 3H, J = 6.6 Hz, 6′-H) indicated that the sugar was also a 6-deoxy-sugar. Its 13C NMR spectrum showed 11 oxygenated carbons at δ 63.3, 66.5, 70.1, 71.9, 74.9, 75.3, 75.9, 77.7, 77.9, 78.1, 85.6 not including the two anomeric carbons. The stereochemistries of the sugars were determined using ROESY and 1D-
TOCSY and by comparison of proton-proton coupling constants and carbon data to those of the sugar portion of digitoxigenin-3-O-[β-D-glucopyranosyl-(1→4)-β-D-boivinopyranosid48 isolated
73 from Corchorus olitorius. Compound 6.5 showed the same HMBC correlations for the first unit
as those depicted in Figure 6.1. The NMR data for compound 6.5 combined with its elemental
composition indicated that it was a new cardenolide glycoside, 5-α-corotoxigenin–3-O-[β-D-
glucosyl-(1→4)-β-D-boivinoside]. It had an IC50 of 0.2 µg/mL against the A2780 cell line.
O
O
HO O O H HO HO O H OH OH H3C O H O H 6.5 OH O
O
HO O H HO HO O H OH OH H3C O H O H OH coroloside
Compound 6.6 was isolated as a white amorphous powder and HRFABMS indicated a
1 molecular formula of C35H52O14. Its H NMR spectrum was almost identical to the spectrum of
6.6; the only difference was the absence of the aldehyde peak at δ 9.98. The 1H NMR spectrum also had signals for two anomeric protons at δ 4.29 (d, J = 7.8 Hz, H-1″) and δ 4.86 (dd, J = 8.7,
3.2 Hz, 1′-H). Its 13C NMR spectrum also confirmed the presence of two anomeric carbons at δ
97.13 (C-1′) and δ 102.10 (C-1″). The presence of a doublet at δ 1.25 (d, 3H, J = 6.6 Hz, 6’-H) also indicated that the sugar was a 6-deoxy-sugar. Its 13C NMR spectrum also showed 11
74 oxygenated carbons at δ 63.0, 66.4, 70.0, 71.9, 74.9, 75.8, 77.8, 78.1, 78.1, 78.5, 86.7 not including the two anomeric carbons. Those peaks confirmed that compounds 6.5 and 6.6 have
the same sugar moieties. The only difference in their 13C NMR spectrum was the absence of the
aldehyde peak at δ 210.4 and the presence of a signal at δ 180.9 for a carboxylic group at C-19.
The NMR data for compound 6.6 combined with its elemental composition indicated that it was
a new cardenolide glycoside.
O
O
O HO HO O HO HO O OH OH H3C O H O H OH
6.6
75 Table 6.1 13C NMR spectra of compounds 6.1-6.3a
Carbon 6.1 6.2 6.3 Lit. data53 Aglycone 5α, 17α-uzarigenin 1 37.0 37.1 38.3 37.5 2 29.6 29.5 31.8 32.3 3 77.4 77.4 73.3 70.6 4 32.1 32.1 35.6 39.1 5 44.1 44.0 45.6 44.9 6 28.6 28.1 28.8 29.2 7 31.6 31.6 29.9 27.6 8 43.8 43.4 42.2 41.5 9 49.6 49.2 49.1 50.2 10 52.8 52.8 36.9 36.0 11 22.9 22.0 21.4 20.0 12 40.4 31.6 30.5 30.8 13 50.7 49.6 51.2 49.3 14 85.8 86.5 87.0 85.2 15 32.8 32.1 31.7 31.5 16 27.9 25.2 25.3 24.8 17 51.9 49.9 49.7 48.9 18 16.1 17.2 18.9 18.6 19 210.4 210.3 12.6 12.4 20 178.2 176.7 176.8 172.9 21 75.3 75.6 75.2 74.1 22 117.9 117.0 116.9 116.6 23 177.2 174.8 175.1 174.1 Sugar I sugars52 1′ 97.5 97.6 97.6 97.0 2′ 35.0 32.1 32.2 32.1 3′ 70.4 80.3 76.5 76.6 4 71.1 70.6 73.4 73.6 5′ 70.2 68.2 70.3 69.3 6′ 16.9 18.6 17.2 17.5 MeO 57.2 57.2 56.6 Sugar II 1″ 102.2 103.3 2″ 74.9 74.7 3″ 78.4 78.5 4″ 71.9 71.9 5″ 78.0 78.5 6″ 63.0 63.1 a) Data for compounds for compounds 6.1-6.3 were obtained in CD3OD and the literature data for 5α,17β-uzarigenin was obtained in C5H5N with TMS as an internal reference. b)Literature data for the of oleandrigenin β-D-glucosyl-β-D-sarmentoside were obtained in CD3OD.
76 Table 6.2: 13C NMR spectra of compounds 6.4-6.6
Carbon 6.4a 6.5b Lit. data48 6.6b Aglycone 5α,17β-uzarigenin55 1 37.3 37.4 37.0 37.8 2 29.6 29.9 29.6 30.8 3 77.3 77.4 77.7 77.8 4 34.4 34.7 32.1 36.8 5 44.2 44.4 44.1 46.0 6 28.9 29.1 28.6 28.9 7 27.9 28 31.6 33.2 8 41.5 41.6 43.8 42.6 9 49.9 50.0 49.6 49.6 10 35.9 36.0 52.8 53.4 11 22.5 21.5 22.9 24.6 12 39.2 39.6 40.4 41.3 13 49.7 49.9 50.7 50.7 14 84.3 84.5 85.8 85.8 15 33.1 33.1 32.8 33.1 16 26.9 27.2 27.9 28.6 17 51.3 51.4 51.9 51.2 18 16.0 16.1 16.1 16.6 19 12.1 12.1 210.4 180.9 20 174.5 174.4 178.2 178.2 21 73.7 73.7 75.3 75.9 22 117.6 117.6 117.9 117.9 23 175.8 175.9 177.2 177.2 Sugar I Lit. data54 Sugars48 1′ 99.9 99.8 97.5 97.6 97.1 2′ 77.3 77.2 35.0 35.2 35.0 3′ 73.1 73.0 66.5 66.6 66.4 4 73.5 73.7 75.9 75.9 75.9 5′ 70.2 70.1 70.1 70.0 70.0 6′ 18.8 18.6 17.2 17.2 17.2 MeO Sugar II 1″ 102.4 102.1 102.3 2″ 74.9 74.9 74.9 3″ 77.9 77.8 78.1 4″ 71.8 71.8 71.9 5″ 78.1 78.0 78.5 6″ 63.0 63.0 63.0 6″ a) in C5D5N; b) CD3OD
77 63 Experimental Section
6.3.1 General Experimental Procedures
Optical rotations were measured with a Perkin-Elmer Model 241 polarimeter. IR and UV spectra were measured on MIDAC M-series FTIR and Shimadzu UV-1201 spectrophotometers, respectively. The NMR spectra were obtained on either a JEOL Eclipse 500 spectrometer or on a
Inova 400 spectrometer. The mass spectra were obtained on a JEOL JMS-HX-110 instrument. A flash chromatograph from Biotage Inc. was used for flash chromatography. HPLC was performed on a Shimadzu LC-10AT instrument using a Varian Dynamax C18 column (250 ´ 10 mm). C-18 SPE columns were obtained from Supelco.
6.3.2 Plant Material
The extract of Roupellina boivinii was received from the National Cancer Institute (NCI) through the ICBG program. The plant was collected on April 21, 1993, in the Diego region of
Antsiranana Provience, Madagascar by D. Harden and Merello from the Missouri Botanical
Garden.
6.3.3 Cytotoxicity Bioassays
The A2780 human ovarian cancer cell line cytotoxicity assay was performed at Virginia
Polytechnic Institute and State University as previously described above.
78 6. 4. 4 Extraction and Isolation
Extract N055899 from Roupellina boivinii (2 g) was partitioned between hexane and 60% aqueous MeOH, and the latter extract was diluted to 50% aqueous MeOH and extracted with
CH2Cl2. All the resulting fractions were evaporated to dryness and tested for their biological activity. The CH2Cl2 and MeOH fractions were the most active with IC50 values of 3.7 and 6.8
µg/mL respectively. The MeOH fraction was then partitioned between BuOH and water, and tested for their activity. The BuOH fraction was the only active fraction with an IC50 value of 4.0
µg/mL. The active fractions of both extracts were separately passed through a short reversed phase chromatography with MeOH/H2O (6/4) as mobile phase, and they were purified further with reversed phase preparative HPLC using MeOH/H2O (6/4) as mobile phase to yield three known compounds 6.1, 6.2, and 6.4 and three new compounds 6.3, 6.5 and 6.6.
79 Roupellina boivinii ( Apocynaceae)
N055899
IC50 = 4.5 2 g
partition hexane/60% aqueous MeOH
partition CH2Cl2/50% aqueous MeOH Hexane Yield 608 mg % 33.4 CH2Cl2 MeOH > 20 Yield 870 mg 634 mg IC50 = % 43.6 31.7
IC µg/mL = 50 3.7 6.8
Short col. C18 MeOH/H O(7/3) 2 500 mg (active fraction)
258 mg used
R. phase chr.MeOH/H2O(7/3) followed by HPLC
6.1 6.2 6.3 6.4 BuOH H2O Yield:(mg) 15 712 10 232 383
IC50 µg/mL = 4.2 20 > 0.22 IC50 µg/mL = 1.5 1.2 0.08 1.1
Only compounds with an IC50 below 3 µg/mL were investigated
6.5 6.6
Combined fractions 1.5 3 Combined fractions with IC above 3 µg/mL with IC5O above 3 µg/mL 5O
Figure 6.4: Bioassay Guided Fractionation of Roupellina boivinii
80 1 Compound 6.1: white amorphous powder (15 mg); H NMR (500 MHz, CD3OD)
(selected chemical shifts); δ 0.81 (s, 3H, H-18), 1.21 (d, 3H, J = 6.6 Hz, H-6′), 2.81 (dd, 1H, J =
5.2, 9.1 Hz, H-17), 3.19 (brd, 1H, J = 3.4 Hz, H-4′), 3.71 (m, 1H, H-3), 3.93 (brd, 1H, J = 3.2 Hz,
H-3′), 3.94 ( dt, 1H, J = 7.8 Hz, H-5′); 4.87 ( dd, 1H, J = 2.0, 10 Hz, H-1′), 4.90, 5.02 (dd, each
1H, J = 2.0, 18.5 Hz, H-21), 5.89 (brs, 1H, H-22), 9.98 (s, 1H, H-19 [COH]); 13C NMR (500
+ MHz, CD3OD, Table 6.1); HR-FABMS m/z found 519.2868 [M+1] ; calcd for C29H43O8,
519.2958.
1 Compound 6.2: white amorphous powder (7 mg); H NMR (500 MHz, CD3OD)
(selected chemical shifts); δ 0.88 (s, 3H, H-18), 1.21 (d, 3H, J = 6.6 Hz, H-6′), 3.23 (dd, 1H, J =
6.8 Hz, H-17), 3.19 (brd, 1H, J = 3.4 Hz, H-4′), 3.71 (m, 1H, H-3), 3.93 (brd, 1H, J = 3.2 Hz, H-
3′), 3.94 ( dt, 1H, J = 7.8 Hz, H-5′); 4.80 ( dd, 1H, J = 1.4, 10 Hz, H-1′), 4.90, 5.02 (dd, each 1H,
J = 2.0, 18.5 Hz, H-21), 5.89 (brs, 1H, H-22), 9.98 (s, 1H, H-19 [COH]); 13C NMR (500 MHz,
+ CD3OD, Table 6.1); HR-FABMS m/z found 555.2928 [M+Na] ; calcd for C30H44NaO8,
555.2934.
1 Compound 6.3: white amorphous powder (12 mg); H NMR (500 MHz, CD3OD)
(selected chemical shifts); δ 0.89 (s, 3H, H-18), 1.26 (d, 3H, J = 6.6 Hz, H-6′), 3.18 (dd, 1H, J =
5.2, 9.1 Hz, H-17), 3.19 (brd, 1H, J = 3.4 Hz, H-4′), 3.66 (dd, 1H, J = 5.5, 12.0 Hz, Hα-6″), 4.03
(m, 1H, H-3), 3.86 (dd, 1H, J = 2.0, 12.0 Hz Hβ-6″), 4.29 (d, 1H, J = 8 Hz, H-1″), 4.85 ( dd, 1H,
J = 2.0, 10 Hz, H-1′), 4.92, 4.96 (dd, each 1H, J = 2.0, 18.5 Hz, H-21), 5.88 (brs, 1H, H-22), 9.98
13 + C NMR (500 MHz, CD3OD, Table 6.1); HR-FABMS m/z found 703.3665 [M+Na] ; calcd for
C36H56NaO12, 703.3669.
81 1 Compound 6.4: white amorphous powder (10 mg); H NMR (500 MHz, CD3OD)
(selected chemical shifts); δ 0.81 (s, 3H, H-18), 1.00 (s, 1H, H-19), 1.68 (d, 3H, J = 6.0 Hz, H-6′),
2.81 (dd, 1H, J = 5.2, 9.1 Hz, H-17), 3.19 (brd, 1H, J = 3.4 Hz, H-4′), 4.03 (m, 1H, H-3), 4.29 (d,
1H, J = 8 Hz, H-1′), ( 4.87 ( dd, 1H, J = 2.0, 10 Hz, H-1′), 4.92, 5.02 (dd, each 1H, J = 2.0, 18.5
13 Hz, H-21), 5.88 (brs, 1H, H-22); C NMR (500 MHz, CD3OD and C5D5N, Table 6.2); HR-
+ FABMS m/z found 543.2927 [M+Na] ; calcd for C30H46NaO8, 543.2934.
1 Compound 6.5: white amorphous powder (1.5 mg); H NMR (500 MHz, CD3OD)
(selected chemical shifts); δ 0.81 (s, 3H, H-18), 1.24 (d, 3H, J = 6.6 Hz, H-6′), 2.81 (dd, 1H, J =
5.2, 9.1 Hz, H-17), 3.19 (brd, 1H, J = 3.4 Hz, H-4′), 3.66 (dd, 1H, J = 5.5, 12.0 Hz, Hα-6″), 4.03
(m, 1H, H-3), 3.87 (dd, 1H, J = 2.0, 12.0 Hz Hβ-6″), 4.29 (d, 1H, J = 8 Hz, H-1″), ( 4.87 ( dd, 1H,
J = 2.0, 10 Hz, H-1′), 4.92, 5.02 (dd, each 1H, J = 2.0, 18.5 Hz, H-21), 5.88 (brs, 1H, H-22) 13C
+ NMR (500 MHz, CD3OD, Table 6.2); HR-FABMS m/z found 703.3327 [M+1] ; calcd for
C35H52NaO13, 703.3305.
1 Compound 6.6: white amorphous powder (3 mg); H NMR (500 MHz, CD3OD)
1 (selected chemical shifts). H NMR (500 MHz, CD3OD) selected chemical shifts; δ 0.81 (s, 3H,
H-18), 1.24 (d, 3H, J = 6.6 Hz, H-6′), 2.81 (dd, 1H, J = 5.2, 9.1 Hz, H-17), 3.19 (brd, 1H, J = 3.4
Hz, H-4′), 3.66 (dd, 1H, J = 5.5, 12.0 Hz, Hα-6″), 4.03 (m, 1H, H-3), 3.87 (dd, 1H, J = 2.0, 12.0
Hz Hβ-6″), 4.29 (d, 1H, J = 8 Hz, H-1″), ( 4.87 ( dd, 1H, J = 2.0, 10 Hz, H-1′), 4.92, 5.02 (dd,
13 each 1H, J = 2.0, 18.5 Hz, H-21), 5.88 (brs, 1H, H-22), C NMR (500 MHz, CD3OD, Table 6.2);
+ HR-FABMS C35H52NaO14 m/z found 719.3251 [M+1] ; calcd for C35H52NaO14, 719.3255.
82 VII. CUCURBITACINS FROM OCTOLEPIS AFF. DIOICA CAPURON
7.1 Introduction
The plant Octolepis aff. dioica Capuron belongs to the family Thymeleaceae. No previous chemical studies of this plant have been reported. The roots, bark wood and leaves of
Octolepis aff. dioica were collected in Madagascar as a part of the ICBG program in order to investigate their potential anticancer activity. Extracts of the roots and leaves only were active against the A2780 cell line.
7.2 Results and Discussion
The EtOH extract of the roots (MG985) and leaves (MG988) of Octolepis aff. dioica
Capuron were subjected separately to solvent partitioning between hexane and aqueous MeOH, and the aqueous MeOH fraction was then extracted with CH2Cl2. All the phases were evaporated under reduced pressure and tested for their biological activities against the A2780 ovarian cancer cell line. The CH2Cl2 fractions of both extracts were the only active fractions, and they were separately purified through reversed phase preparative HPLC using MeOH/H2O (6/4) as mobile phase to yield three compounds, 7.1 and 7.2 from MG985, and 7.3 and 7.2 from MG988, identified as cucurbitacins D, I, and K respectively.
Compound 7.1 was isolated as an amorphous substance. Its HRFABMS indicated a
1 molecular formula of C30H44O7. Its H NMR spectrum revealed 8 methyl singlets at δ 0.97, 1.09,
1.30, 1.34, 1.34, 1.36, 1.36, 1.40; some of the peaks were overlapped and were identified by integration. The spectrum also showed a doublet and broad doublet δ 2.71 (d, J = 14 Hz, Hα-12) and 3.29 (bd, J = 14 Hz, Hβ-12), a broad triplet at δ 4.34 (bt, J = 7 Hz, H-16), and a doublet of
83 doublets at δ 4.44 (dd, J = 12, 6 Hz, H-2). Finally, signals for protons on two double bonds were
observed at δ 5.78 (m, H-6), 6.62 (d, J = 15 Hz, H-23) and 7.14 (d, J = 15, H-24).
The 13C NMR spectrum of compound 7.1 indicated the presence of 30 carbons with signals for three ketone peaks at δ 202.6, 212.4 and 213.1 assigned to C-22, C-3 and C-11 respectively, 4 oxygenated carbons at δ 71.2, 71.5, 71.7, 78.2 assigned to C-25, C-16, C-2, and
C-20 respectively, and other peaks. This spectrum also confirmed the presence of two double bonds at δ 119.1, 120.3, 140.6, and 156.0 assigned to C-23, C-6, C-5 and C-24 respectively. Its
DEPT spectrum revealed that the skeleton was made of 8 methyls, four methylenes, 7 methines, and 11 quaternary carbons. The NMR data for compound 7.1 combined with its elemental composition and a literature search indicated that it was most probably the previously reported cucurbitacin D.57,58 A comparison of its NMR data with the literature data of cucurbitacin D57,59
indicated that compound 7.1 was indeed cucurbitacin D. It had an IC50 of 0.05 µg/mL against the
A2780 cell line.
O 21 OH 24 26 18 OH O 11 H 23 17 27 1 H H OH HO 15 3 19 30 O 6 28 29 7.1 (Cucurbitacin D)
57 Velde, V. V.; Lavie, D. 13C NMR spectroscopy of cucurbitacins. Tetrahedron 1983, 30, 317-321 58 Jayaprakasam, B.; Seeram, N. P.; Nair, M. G.; Anticancer and anti-inflammatory activities of cucurbitacins, Cancer Lett., 2003, 189, 11-16 59 Che, C-T; Fang, X.; Phoebe, C. H.; Kinghorn, A. D.; Farnsworth, N.R. High-field 1H NMR spectral analysis of some cucurbitacins, J. Nat. Prod. 1985, 48, 429-434
84 Compound 7.2 was also isolated as an amorphous substance. Its HRFABMS indicated a
1 molecular formula of C30H42O7. Its H NMR spectrum was identical to the spectrum of
compound 7.1 except for the presence of signals for one extra double bond at δ 5.97 (d, J = 3 Hz,
H-1). The 13C NMR spectrum also indicated the presence of 30 carbons, including three ketone peaks at δ 198.7, 212.9 and 202.7 assigned to C-3, C-11, and C-22 respectively, and three oxygenated carbons at δ 71.2, 71.6, 78.1 assigned to C-25, C-16, and C-20 respectively. Its 13C
NMR spectrum also confirmed the presence of three double bonds at δ 115.0, 119.1, 120.7,
137.0, 144.6, and 155.9 assigned to C-1, C-23, C-6, C-5, C-2 and C-24 respectively. The NMR data for compound 7.2 combined with its elemental composition and literature search indicated that it was most probably the previously reported cucurbitacin I.57,59 A comparison of its NMR data with the literature data of cucurbitacin I57,59 indicated that compound 7.2 was indeed cucurbitacin I. It had an IC50 of 0.5 µg/mL against the A2780 cell line.
OH O OH O H H H OH HO
O 7.2 (Cucurbitacin I)
Compound 7.3 was also isolated as an amorphous substance. Its HRFABMS indicated a
1 molecular formula of C30H43O8. Its H NMR spectrum was identical to the spectrum of compound 7.2 except for the presence of three doublets of doublets at δ 2.98 (dd, J = 16.1, 1.1
85 Hz, H-23), δ 2.68 (dd, J = 16.1, 9.8 Hz, H-23), and δ 3.91 (dd, J = 9.5, 1.7 Hz, H-24) and for the absence of two doublets at δ 6.62 (H-23) and 7.14 (H-24), indicating the absence of double bond at position C23/24 and the presence of a hydroxyl group at C-24. The 13C NMR spectrum also indicated the presence of 30 carbons, including three carbonyl peaks at δ 198.7, 212.9 and 215.5 assigned to C-3 and C-11 and C-22 respectively, and also peaks for four oxygenated carbons at δ
71.1, 72.3, 74.3, 79.4 assigned to C-16, C-25, C-24, and C-20 respectively. Its 13C NMR spectrum also confirmed the presence of two double bonds at δ 114.9, 120.7, 136.9, 144.6 assigned to C-1, C-6, C-5, C-2 respectively. The NMR data for compound 7.3 combined with its elemental composition indicated that it was most probably the known cucurbitacin K. 60 A comparison of its NMR data with the literature data60 of cucurbitacin K indicated that compound
7.3 was indeed cucurbitacin K. It had an IC50 of 2.0 µg/mL against the A2780 cell line.
OH O OH OH O H H H OH HO 7.3 (Cucurbitacin K) O
60 Kanchanapoom, T. ; Kasai, R. ; Yamasaki, K. Cucurbitane, hexanorcucurbitane and octanorcucurbitane glycosides from fruits of Trichosantes tricuspidata, Phytochem. 2002, 59, 215-228
86 Table 7.1:13C NMR data of compounds 7.1, 7.2 and 7.3
Carbon 7.1 Lit.57,59 7.2 Lit.57,59 7.3 Lit.60 1 36.1 36.0 114.9 115.0 115.0 114.9 2 71.6 71.7 144 144.6 144.7 144.6 3 212.3 212.4 198.6 198.7 198.9 198.7 4 50.3 50.3 47.6 47.6 47.4 47.6 5 140.5 140.6 136.9 137.0 137.2 136.9 6 120.4 120.3 120.6 120.7 120.5 120.7 7 24.0 24.0 23.6 23.6 23.6 23.6 8 42.5 42.5 41.6 41.6 41.5 41.6 9 48.5 48.4 48.7 48.8 50.7 50.7 10 33.8 33.8 34.6 34.7 34.6 34.7 11 213.0 213.1 212.8 212.9 212.8 212.9 12 48.7 48.8 48.8 48.8 48.8 48.8 13 48.3 48.3 48.2 48.3 48.7 48.8 14 50.8 50.9 50.7 50.8 48.5 48.4 15 45.6 45.6 45.7 45.7 45.7 45.7 16 71.5 71.5 71.6 71.6 71.2 71.1 17 57.3 57.3 57.3 57.4 57.5 57.5 18 20.1 20.1 19.9 20.0 19.9 19.8 19 19.2 19.3 18.5 18.6 18.4 18.3 20 78.3 78.2 78.1 78.1 79.4 79.4 21 23.9 24.0 23.9 24.0 24.2 24.3 22 202.6 202.7 202.6 202.7 215.4 215.5 23 119.1 119.1 119.1 119.0 38.2 38.3 24 155.9 156.0 155.9 155.9 74.3 74.3 25 71.2 71.2 71.2 71.2 72.2 72.3 26 28.9 28.9 28.9 29.0 25.7 25.7 27 29.6 29.6 29.6 29.6 24.7 24.7 28 21.2 21.3 20.0 20.1 20.0 20.1 29 29.3 29.4 27.8 27.9 27.9 27.9 30 20.0 20.1 20.0 20.1 20.1 20.2
87 7.3 Experimental Section
7.3.1 General Experimental Procedures
The NMR spectra were obtained on either a JEOL Eclipse 500 spectrometer or on a
Inova 400 spectrometer. The mass spectra were obtained on a JEOL JMS-HX-110 instrument. A flash chromatograph from Biotage Inc. was used for flash chromatography. HPLC was performed on a Shimadzu LC-10AT instrument using a Varian Dynamax C18 column (250 ´ 10 mm). C-18 SPE columns were obtained from Supelco.
7.3.2 Cytotoxicity Bioassays
The A2780 human ovarian cancer cell line cytotoxicity assay was performed at Virginia
Polytechnic Institute and State University as previously described.
7.3.3 Plant Material
The extracts, root, wood, bark and leaves, of Octolepis aff. dioica Capuron
(Thymeleaceae) were collected by N. M. Andrianjafy and his assistants from the rainforest of
Toamasina, Madagascar, in the vicinity of Zahamena on October 4, 2001. The roots, bark, leaves and wood of the dried plant material were extracted with EtOH separately to yield extracts
MG985 (roots), MG986 (bark), MG987 (wood) and MG988 (leaves). Only MG985 and MG988 were active against the A2780 cell line, with an IC50 value of 16 and 23 µg/mL respectively.
7.3.4 Extraction and Isolation
Extracts MG985 (2.24 g) and MG988 (0.73 g) from Octolepis aff. dioica Capuron
(Thymelaeaceae) were partitioned between hexane and 60% aqueous MeOH separately and the
88 latter extract was diluted to 50% aqueous MeOH and extracted with CH2Cl2 (Figure 7a and 7b).
All the resulting fractions were evaporated to dryness and tested for their biological activity; only the CH2Cl2 fractions of both extracts were active. The active fractions of both extracts (MG985,
MG988) were separately fractionated further through normal phase flash chromatography with
CH2Cl2/MeOH (9/1) as mobile phase, and the most active fractions were purified further with reversed phase analytical HPLC using MeOH/H2O (6/4) as mobile phase to yield three compounds 7.1 (5mg), 7.2 (4.5mg), 7.3 (3.3 mg), known as cucurbitacin D, I, and K
1 Compound 7.1: Colorless amorphous substance (5 mg); H NMR (500 MHz, CDCl3): δ
0.97 (s, H-18), 1.09 (s, H-30), 1.29 (s, H-29), 1.33 (s, H-28), 1.34 (s, H-26), 1.35 (s, H-27), 1.36
(s, H-21), 1.38 (s, H-28), 1.40 (s, H-19), 1.87 (m, H-15), 1.99 (dd, J = 18.6, 7.5 Hz, 2.25 (m, Hα-
7), 2.37 (m, H-8), 2.54 (d, J = 7.0 Hz, H-17), 2.72 (d, J = 14.8 Hz, Hβ-12), 3.29 (brd, J = 14.9 Hz,
Hα-12), 2.85 (brd, J = 13.4 Hz, H-10), 4.36 (t, J = 7.2 Hz, H-16),4.43 (dd, J = 12,6), 5.75 (brs, H-
6); 6.61 (d, J = 15 Hz, H-23), 7.14 (d, J = 14.9 Hz, H-24). 13C NMR (see Table 7.1). HR-
+ FABMS found m/z 517.3163 [M+1] ; calcd for C30H45O7, 517.3165.
1 Compound 7.2: Colorless amorphous powder (5 mg); H NMR (500 MHz, CDCl3) δ
1.00 (s, H-18), 1.04 (s, H-30), 1.26 (s, H-29), 1.36 (s, H-26), 1.35 (s, H-27), 1.38 (s, H-21), 1.38
(s, H-28), 1.41 (s, H-19), 1.87 (m, H-15), 1.99 (dd, J = 18.6, 7.5 Hz, 2.25 (m, Hα-7), 2.37 (m, H-
8), 2.54 (d, J = 7.0 Hz, H-17), 2.80 (d, J = 14.8 Hz, Hβ-12), 3.15 (brd, J = 14.9 Hz, Hα-12), 2.85
(brd, J = 13.4 Hz, H-10), 4.38 (t, J = 7.2 Hz, H-16), 5.75 (brs, H-6), 5.96 (d, J = 2.8 Hz, H-1);
6.61 (d, J = 15 Hz, H-23), 7.12 (d, J = 14.9 Hz, H-24). 13C NMR (see Table 7.1). HR-FABMS
+ found m/z 515.3006 [M+1] ; calcd for C30H43O7, 515.3008.
89 1 Compound 7.3: Colorless amorphous powder (3.3 mg); H NMR (500 MHz, C5D5N) δ
0.99 (s, H-30), 1.01 (s, H-18), 1.19 (s, H-26), 1.22 (s, H-27), 1.24 (s, H-29), 1.34 (s, H-28), 1.38
(s, H-21), 1.41 (s, H-19), 1.87 (m, H-15), 2.01 (m, H-7), 2.37 (m, H-8), 2.54 (d, J = 7.0 Hz, H-
17), 2.67 (dd, J = 16.0, 9.8 Hz, Hβ-23), 2.97 (dd, J = 16.0, 1.2 Hz, Hα-23), 2.69 (d, J = 14.3 Hz,
Hβ-12), 3.20 (d, J = 14.1 Hz, Hα-12), 3.49 (brs, H-10), 3.90 (dd, J = 9.5, 1.6 Hz, H-24), 4.38 (t, J
= 7.4 Hz, H-16), 5.75 (brs, H-6), 5.94 (d, J = 2.6 Hz, H-1); 13C NMR (see Table 7.1). HR-
+ FABMS found 533.2977 m/z [M+1] ; calcd for C30H45O8, 533.2879.
90 Octolepis aff. dioica Capuron (Thymeleaceae) Samples MG985 Available: 3g Used: 2.5g Remaining: 0.7g IC50 (µg/mL ) = 16 2.24g
partition hexane/60% aqueous MeOH
partition CH2Cl2/50% aqueous MeOH
Hexane Yield 20.7
% 0.9 CH2Cl2 MeOH Yield 325.3 1.9 IC50 (µg/mL ) < 20 % 14.5 84.6
IC50 (µg/mL ) = 3.7 Flash chrom.normal phase CHCl3/MeOH: 9/1
1 2 3 4 6 7 14.4 Yield 34 30.1 21 85.6 120.
% 4.4 10.4 9.2 4.1 26.3 36.9
IC50 (µg/mL ) 12.5 3.6 0.8 6.8 16.8 NA
Reverse phase HPLC MeOH/H2O: 6/4
3-1 3-2 3-3
Yield 15.3 5.0 4.5
IC50 = < 20 0.5 0.5 7.17.2
Figure 7.1: Bioassay guided fractionation of Octolepis aff. dioica Capuron (Thymeleaceae)
91 Octolepis aff. dioica Capuron (Thymeleaceae) MG988 Samples Available:7g Used:727.3 mg Remaining:5.8 g
IC50(µg/mL ) = 23 727.3 mg
partition hexane/60% aqueous MeOH
partition CH2Cl2/50% aqueous MeOH
Hexane Yield 18.7 CH2Cl2 MeOH % 2.6 Yield 57mg651.6mg IC =15.6 50 % 7.8 89.6 < 20 IC50(µg/mL ) 3.8
Flash chrom.C18 MeoH/H20: 6/4
1 4 5 Yield 34.7 mg 11.5 mg 6.1mg
IC50 < 20 IC50(µg/mL ) 2.5 < 20
HPLC C18 MeoH/H20: 6/4
Yield 3.5 3.3 mg2.5 mg
IC50(µg/mL ) < 20 2.00.5
7.37.2
Figure 7.2: Bioassay guided fractionation of Octolepis aff. dioica Capuron (Thymelaeaceae)
92 VIII. OTHER PLANTS STUDIED, BUT DROPPED
Elephantopus carolinianus (extract # 980319E, IC50 = 5.0 μg/mL). The extract was received from Suriname. This plant has been widely studied and is known to contain sesquiterpene lactones which are known to be cytotoxic. The plant was reinvestigated in the hope that new bioactive compounds would be isolated, but this turned out not to be the case. After partition, fractionation and further purified, the same known compounds as had previously been found in this plant were isolated. They were identified as the sesquiterperne lactones 8.1-8.3 by comparison of their spectroscopic data with literature data.61
O O
O
HO IC50 = 1.8 µg/mL O
O O O O O O O
O O O O O O
O O
IC = 2.3 µg/mL IC50 = 2.3 µg/mL 50
Siparuna guianesis (aubl.) (Monimiaceae) (Extract # 970540, IC50 = 17.5 μg/mL),
Lantana camara (Extract # 980334 E/M, IC50 = 17 μg/mL), Turraea species (Extract # MG
0234/1675, IC50 = 40 μg/mL). These plant extracts were partitioned, fractionated and tested.
61 Lee, K.H; Cowherd, C. M.; Wolo, M. T. Antitumor agents. XV: Deoxyelephantopin, an antitumor principle from Elephantopus carolinianus Wild. J. Pharm. Sc. 1975, 64, 1572-3
93 None of the fractions tested showed any improvement in their cytotoxic activities, so these extracts were dropped.
Gomphocarpus fruticosus (Apocynaceae) (Extract # MG1222, IC50 = 1.8 μg/mL and
MG1221, IC50 = 5.9 μg/mL). The extract was partitioned and fractionated. The CH2Cl2 fraction
1 had an IC50 = 0.4 μg/mL. After further purification, a H NMR spectrum revealed that all the semi-pure compounds, which had IC50 values of approximately 0.2 μg/mL, were cardenolide glycosides. Cardenolide glycosides such as those isolated from Roupellina boivinii, are of low interest as anticancer agents because of their toxicity, and so it was decided not to pursue the isolation and structural elucidation of these compounds.
Croton sp. (Euphorbiaceae) (Extract MG290), Bixa orellana L. (Extract # 980291E) and
Asteraceae sp. (Extract # 980285M). All these plants were investigated for their potential antimalarial activity. The crude extract of Asteraceae sp. was originally tested for antimalarial activity at the Walter Reed Army Institute of Research and retested at the “Instituto de Medicina
Tropical y Ciencias de la Salud” in Panama where it had an IC50 of 7 µg/mL against Plasmodium falciparum in both assays. Croton sp and Bixa orellana were both tested at the “Instituto de
Medicina Tropical y Ciencias de la Salud” in Panama, and only Bixa orellana was active with an
IC50 of 11 µg/mL. The two active extracts were partitioned, fractionated and sent to our collaborators in Madagascar to be tested in a new assay. None of the fractions were active in that assay, and work on the extracts was thus deferred until consistent assay results could be obtained.
94 PART II
A SYNTHETIC APPROACH TO LUCILACTAENE AND ITS ANALOGUES
I Introduction
1.1 The Cell Cycle as a Target for Anticancer Drugs
Cancer can be caused by both external factors (chemicals, radiation, tobacco and
infectious organisms) and internal factors (mutations, hormones, and immunological conditions).
Those two factors can promote carcinogenesis by destroying the cell’s control mechanisms. The
study of cell cycle pathways as molecular targets led to the identification of cyclin-dependent
kinases that control cell cycle mechanisms. Cell cycle events are divided into four phases known
62, 63 as G1, S, G2, and M (Figure II.1). The G1 checkpoint is the phase in which the cell prepares for DNA synthesis and ensures that DNA damage is repaired before the S phase, which is the stage where DNA is replicated. The G2 checkpoint ensures DNA integrity before the cell enters mitosis, and this M phase leads to the formation of two daughter cells. Therefore the proper function of the G1 and G2 checkpoints is crucial for correct cell division.
62Buolamwini, J.K. Cell cycle molecular targets in novel anticancer drug discovery. Curr. Pharm. Design 2000, 6, 379-392 63 Lundberg, A. S; Weinberg, R. A. Control of the cell cycle and Apoptosis. Eur. J. Cancer 1999, 35, 531-539
95 cdk1
Cyclin B G0 cdk1 G Cyclin A 2 M pRb
E2F cdk4/cdk6
S Cyclin D G1
cdk2 cdk2 pRb E2F Cyclin E Cyclin A P P
Figure II.1: Cell cycle62, 63
1.2 Cell Cycle Inhibitors
Cell-based screening approaches have led to the discovery of several leading compounds as candidates for cancer therapy. Among these compounds, flavopiridol, 64 UCN-01 (7- hydroxystaurosporine)4 and E7070, a chloroindolyl sulfonamide,65 are all known to inhibit cell proliferation and are well advanced in clinical development. Flavopiridol inhibits all cyclin- dependent kinases (CDKs) that control the cell-cycle evolution by docking in the ATP-binding site and inducing cell apoptosis. UCN-01 inhibits the Chk1/2 kinases and cell proliferation by acting on Ca2+-dependent PKC isozymes; it also causes apoptosis by a still unknown mechanism.
64 Senderowicz,A. M.. The cell cycle as a target for cancer therapy: basic and clinical findings with the small molecule inhibitors flavopiridol and UCN-01. The Oncologist, 2002, 7(Suppl 3), 12-19 65 Raymond, E., Huinink, B. W. W. , Taieb, J, Beijnen, J.H. , Faivre, S, Wanders, Ravic, J.M., Fumoleau , P., Armand, J.P. and Schellens, J. H. M. Phase I and pharmacokinetic study of E7070, a novel chloroindolyl sulfonamide cell cycle inhibitor, administered as one hour infusion every three weeks in patients with advanced cancer. J. Clin. Oncol., 2002, 20, 3508-3521
96 E7070 induces both G1-S and G2-M arrest in HCT-116 human colon cancer cells. At higher concentrations E7070 regulates p53 and p21 and as a result causes cell death. Olomoucine is another interesting compound that inhibits the proliferation of human cancer cell lines such as breast and pancreatic cancers as well as lymphomas. Olomoucine arrests lymphoma cells in the
2 G1 and G2 phases by inhibiting cyclin E/CDK2 and cyclin B/CDK1.
H OH O O N OH Cl
HO O OH N N O H CH N 3
CH3 OCH3 NHCH3
Flavopiridol UCN-01: 7-hydroxystaurosporine
HN
N N O O Cl N S S NH HN N N H 2 NH O O CH3
OH E7070 : N-(3-chloro-7-indolyl)-1,4- benzenedisulfonamide Olomoucine
1.3 p53 Tumor Suppressor Gene
Researchers from different scientific disciplines have combined their efforts in order to understand cancer. Within the last decade, genomics research has identified many new genes that play crucial roles in cancer development and progression. Among these genes, the p53 gene and its protein product have been the subject of many studies. More than 50 % of human cancers
97 contain mutations in this gene.66 The p53 tumor-suppressor gene controls cell life and death.
Some researchers call this gene the “cellular gatekeeper” for growth and division, and others
compare it to the brake of a car which controls its speed. The normal tumor-suppressor genes
play the role of brakes to the cycle of cell growth, DNA replication and division into two new
cells. A mutation in p53 or a failure in its function leads to cells that grow out of control, a
defining feature of cancer cells. The p53 protein stimulates the expression of p21WAF1/CiP1, an
inhibitor of cyclin-dependent kinases (CDKs) which are key regulators of the cell cycle.
1.4 Lucilactaene, a New Cell Cycle Inhibitor
Lucilactaene is an unusual pentaene produced by the fungal strain RK97-94 identified as
a Fusarium sp. It was isolated using a bioassay based on the H1299/tsp53 cell line.67 H1299 cells
(human non-small lung cancer cells) have the p53 gene mutated, and lucilactaene was found to
inhibit cell cycle progression and to stop cell division at the G1 phase.
Lucilactaene is a small molecule that induces cell cycle arrest by inhibiting the mutant
p53 gene and, as a consequence, it allows the mutant gene to maintain its active form which is to
control the cell cycle. Therefore such a molecule that can control the cell cycle might be an
excellent candidate for the development of an anticancer drug. The fungal strain used was
isolated from the leaf of an unidentified plant collected in Japan; this strain was successfully
cultivated by fermentation at 28 ºC for 96 hours.7 The mycelia (mass of filaments) produced by
the culture (30 liters) were extracted with acetone. The crude extract was partitioned between
aqueous solution and ethyl acetate. The oily residue from the organic phase was purified by silica
66 Vogelstein, B., Lane, D., and Levine, A. J. Surfing the p53 network. Nature, 2000, 408, 307-310 67 Kakeya, H., Kageyama, S. I., Nie, L., Onose, R., Okada, G. Beppu, T. Norbury, C. J, Osada, H. Lucilactaene, a new cell cycle inhibitor in p53-transfected cancer cells, produced by a Fusarium sp. J. Antibiot.2001, 54, 850-854
98 gel column chromatography. The pure compound (50 mg) was obtained as a pale yellow
amorphous solid after further purification on a HPLC reverse-phase column.
CO2CH3 O H O O NH 1 OH
CO2CH3 O O O NH HO OH NG-391
The structure of lucilactaene 1 is made up of a tetrahydrofuran ring fused with a lactam and a side chain with a long all-E conjugated double bond system. The structure activity relationships of the compound have not been established, but the bicyclic ring seems to be necessary for its activity. This conclusion is drawn by comparing its structure to NG-391, which was an inactive second compound isolated from the same Fusarium sp. NG-391 was later found
to be a neuronal cell-protecting molecule. Based on the unique bioactivity of lucilactaene 1, the
compound was selected for synthesis, along with analogues derived by replacing the methyl
group at C2 with different groups such as phenyl, thiazole, furan and thiophene. The synthetic
approach selected could also yield 13-epi isomers of lucilactaene and also lactone intermediates,
and their activity could thus be evaluated and compared to the activity of the parent compound,
lucilactaene.
99
CO2CH3 CO2CH3 O O H H H H R R O O O O
NH NH 1'( 13-epi isomer) OH 1 OH
R = CH3 , lucilactaene
Analogues: R = N O S S
CO2CH3 CO2CH3 O O H H H H R R O O O O O O 1b' 1a
Lactone lucilactaene analogues
100 II Results and Discussion
2.1 Retrosynthetic analysis
The total synthesis of lucilactaene could be achieved using the disconnection approach shown in Scheme 1 below.
Scheme 1: Retrosynthesis of lucilactaene
O OCH3 O H O O NH OH
O OCH3 O H O NH2 I O OH
H O OCH3 O R P H O + 3 O X III II O OR
O O OSi O O + O OR OCH3 VIV
101 Lucilactaene could be obtained by oxidation of the alcohol I to a ketone which would cyclize spontaneously. The intermediate I could be assembled based on the disconnection shown in the
Scheme 1. The known fragment II was synthesized as previously reported.68
2.2. Synthetic Aproach
2.2.1. Fragment II
CO2CH3 R3P R
II Compound 5 was synthesized according to the known procedure described below in Scheme 2.68
Scheme 2. Synthesis of compound 5
OO OO a b HO I EtO OEt EtO OEt CHI2 O 2 Diethyl methyl malonate 1
c
e d TBDPSO SnMe3 TBDPSO I HO I 5 4 3
a) CHI3, NaH, Et2O, reflux 18h; b) KOH,EtOH-H2O (3:1) reflux, 24h; c) LiAlH4, THF, 0 ºC; d) TBDPSCl, imidazole, DMF, rt 18 h.; e) t-BuLi, THF, -78 ºC, then Me SnCl 3
68 Marumoto, S. ; Kogen, H; Naruto, S. Asymmetric total synthesis of epolactaene. Part 2: Introduction of the side chain and synthesis of (+)-epolactaene and its enantiomer Tetrahedron, 55, 1999, 7145-7156
102 Commercially available diethyl methylmalonate was treated with sodium hydride in dry diethyl ether, followed by the addition of iodoform. The resulting mixture was refluxed for 24h under nitrogen and worked up to give diiodide 1.69 Compound 1 was then refluxed in ethanol- water in the presence of KOH to yield compound 2. Reduction of the obtained acid 2 with
LiAlH4 yielded the allylic alcohol 3. The allylic alcohol was protected with tert- butyldiphenylsilyl chloride (TBDPS) to afford compound 4.68 The protected vinyl iodide 4 was transformed to the corresponding vinyllithium by halogen-lithium exchange with tert- butyllithium in THF at -78 ºC, followed by treatment with trimethyltin chloride to afford the vinylstannane 5.68
The α,β-unsaturated esters 6 were prepared according to the known literature procedure as described in Scheme 3 below.70 Compounds 6a and 6b were prepared by direct bromination of the ylide, Ph3P=CHCO2CH3, with N-bromosuccinimide, and the Wittig reaction of the resulting bromoylide with the different aldehydes in the presence of potassium carbonate.
Scheme 3. Synthesis of α,β-unsaturated ester 6
R O O 0 Br OCH3 1) CH Cl , -20 C 2 2 Ph3P + N Br OH O OCH3 O 2) K2CO3 , R 6a/b
6a R = CH3 6b R = 4-(2-methyl thiazolyl)
69 Baker, R. and Castro, J.L. Total synthesis of (+) - macbecin. J. Chem. Soc. Perkin Trans.1, 1990, 47-65 70 Kayser, M. M., Zhu, J. ; Hooper D. L. Stabilized haloylides: Synthesis and reactivity. Can. J. Chem. 1997, 75, 1315-1321
103 Scheme 4. Synthesis of fragment II
O OCH3 O OCH3 a + TBDPSO TBDPSO SnMe3 Br R R 6a/b 7a/b 5
b
O OCH O OCH3 3 O OCH3 HO Bu3P Br Br- d c R R R 9a/b 8a/b 10a/b (Fragment II)
a) [Pd(PPh3)4, CuCl, LiCl, DMSO/THF, 1/1]; b) TBAF; c) CBr4,Ph3P, d) n-Bu3P
The vinylstannane 5 was coupled with the different compounds 6a and 6b by a modified
Stille coupling procedure [Pd(PPh3)4, CuCl, LiCl, DMSO/THF] to yield dienes 7a and 7b (66 to
80% yield depending on the R group) in a stereoselective fashion.71 Compound 7a is a known
fragment,68 and several attempts to cross-couple vinylstannane and compounds 6a and 6b under
68 the reported conditions [Pd(PPh3)4, toluene, reflux] yielded products in less than 20 % yield
although a 47 % yield was reported. The silyl groups of the dienes 7a and 7b were removed with
tetrabutylammonium fluoride (TBAF) to provide primary alcohols 8a and 8b.68 Compounds 8a
and 8b were transformed to bromides 9a and 9b using carbon tetrabromide (CBr4) and
72 triphenylphosphine in CH2Cl2, and the resulting bromides, 9a and 9b were treated with
tributylphosphine to give phosphonium salts 10a and 10b.68
71 Fuma, H.; Kainuma, N.; Tachibana, K.; and Sasaki, M. Total synthesis of (-)-gambierol, J. Am. Chem. Soc. 2002, 124, 14983-14992 72 Sugiyama, H.; Yokokawa, F. and Shioiri. Total synthesis of mycothiazole, a polyketide heterocycle from marine sponges, Tetrahedron, 2003, 59, 6579-6593
104 2.2.2 Fragment IV
O O Si OMe
O Fragment IV
Fragment IV was synthesized as described below in Scheme 5. Ethyl hydroxycrotonate
(12) was prepared from commercially available monoethyl fumarate 11 according to a slightly modified known procedure.73 The resulting alcohol was protected with tert-butyldimethylsilyl- chloride to yield compound 13, which was reduced with DIBAL-H to afford compound 14. The resulting alcohol was oxidized to aldehyde 15 using the Swern oxidation procedure74 to give compound 15, which was then coupled with the commercially available Wittig reagent,
(carbethoxyethylidene)-triphenylphosphorane, to yield diene 16. Compound 16 was transformed to the Weinreb amide 17 by following the known procedure, using N,O-dimethyl-hydroxylamine hydrochloride and isopropyl magnesium chloride in THF at -20 ºC.75,76 The resulting Weinreb amide and methyl acetate were coupled at -78 ºC using LHMDS as base, a known procedure,77 to give the β-keto methyl ester, compound 18.
73 Organic Syntheses, 1986, 64, 104-107 74 Smith III, A. B.; Beauchamp, T. J.; LaMarche, M. J. ; Kaunfman, M. D.; Qiu, J.; Arimoto, H.; Jones, D. R.; Kobayashi, K. Evolution of a gram-scale synthesis of (+)-discodermolide, J. Am. Chem. Soc, 2000, 122, 8654-8664 75 Evans, D. A.; Rajapakse, H. A.; Stenkamp,D. Asymmetric syntheses of pectenotoxins-4 and -8. Part I: Synthesis of the C1-C19 subunit. Angew. Chem. Int. Ed. 2002, 4, 4569-4573 76 Williams, M. J.; Jobson, r. b.; Yasuda, N.; Marchesini, G. A new general method for preparation of N-methoxy-N- methylamides. Application in direct conversion of an ester to a ketone. Tetrahedron Lett. 1995, 36(31), 5461-5464 77 Turner, J. A.; Jacks, W. S. Acylation of ester enolates by N-methoxy-N-methylamides: an effective synthesis of β- keto esters, J. Org. Chem. 1989, 54, 4229-4231
105 Scheme 5 Synthesis of fragment IV
O OEt a OEt b OEt HO HO t-BuMe2SiO O O O 11 12 13
c
OEt O OH t-BuMe2SiO t-BuMe2SiO e t-BuMe2SiO O H d 16 15 14
f
OMe NMe g OMe t-BuMe2SiO t-BuMe2SiO O O O
17 18 Fragment IV
a) BH3 THF, -10 ºC to rt; b)TBDMSCl, imidazole, DMF, rt; c) DIBAL, toluene, -78 ºC d) DMSO,oxalyl chloride, i-Pr2NEt; e) Ph3P=CCO2Et(CH3), CH2Cl2, reflux; f) Me(MeO)NH/HCl, i-PrMgCl, THF g) n-BuLi, methylacetate, THF, -78 ºC
106 2.2.3. Fragment V
O O O
O Ph Ph Ph Fragment V
Fragment V was obtained according to Scheme 6 described below. (S)-Malic acid was converted to compound 19 with 2,2-dimethoxypropane in the presence of PPTs. The resulting protected acid was reduced to the alcohol 20 using BH3•THF. The unstable product was then heated in the presence of PPTs to afford (S)-3-hydroxybutyrolactone 21.78 Treatment of (S)-3- hydroxybutyrolactone 21 with trityl chloride and DBU as base in CH2Cl2 yielded (S)-3- trityloxybutyrolactone 22. Reduction of compound 22 with DIBAL-H afforded 23 as a mixture of epimers. This was treated with acetic anhydride in pyridine79 to yield exclusively trans trityl- acetoxy product 24, fragment V.
78 Green D. L. C.; Kiddle, J. J.; Thompson, C. M. Stereochemistry of remote dianion addition to imines. Application to the synthesis of (1S, 8aS)-1-hydroxyindolizidine, Tetrahedron, 1995, 51, 2865-2874 79 Corey, E. J.; Kania, R. S. First enantioselective total synthesis of a naturally occurring dolabellane. Revision of absolute configuration, J. Am. Chem. Soc. 1996, 118, 1229-1230
107 Scheme 6 Synthesis of fragment V
O OH O O O O HO O acb OH OHO O O O OH O OH S-malic acid 19 20 21
d O O O O O O fOH e O OTr OTr Ph Ph 24 23 Ph
o o 22 a) 2, 2 Dimethoxy-propane, PPTs, rt; b) BH3/THF, -78 C to rt; c) PPTs, toluene, 65 C o d) triphenylmethane chloride, DBU, CH2Cl2, rt ; e) DIBAL, toluene, -78 C; f) pyridine, Ac2O, rt
2.2.4 Fragment III
H O 3 O H 7 O 2 O 2' 1 3' OCH3 O Ph Ph Fragment III Ph
Fragment III was assembled through the synthetic steps described in Scheme 7 below.
The silyl enol ether 25 was prepared by treating fragment IV (18) with LiHMDS at -78 ºC, and then trapping the resulting enol formed with TMSCl. Compound 24 was coupled with the silyl enol ether 25 using a well known C-glycosidation method to form C-C bonds at the anomeric
108 center of carbohydrates. Silver (I) triflate (AgOTf), boron trifluoride ether complex (BF3/Et2O)
and bismuth tribromide were tried first and gave little or no product. The best results were
achieved when the Lewis acid was changed to Sc(OTf)3. These conditions afforded a mixture of four separable diastereomers 26 with the trans-substituted products as the major product.
Sc(OTf)3 has been reported to be a good catalyst for the imino-version of a C-glycosylation reaction and to give diastereoselective substituted products.80
Scheme 7.1 Coupling Fragment V (24) and IV (24)
O TMSO O O O O OTBS OTBS H OTBS + O O O H Sc(OTf)3 O O OTr OCH CH2Cl2 + 3 OCH3 OCH OTr 3 24 25 OTr 26 27
a
H O O O OH H H O O O O a) TBAF; b) Dess-Martin b periodinane OCH3 OCH3 OTr OTr 29a/b 28a/b
Diastereoselective nucleophilic substitution products were also observed in this reaction.
In this reaction, scandium triflate (Sc(OTf)3) activated the acetoxy carbon to the oxonium ion 24′.
80 Okitsu, O.; Suzuki, R.; Kobayashi, S.; Efficient synthesis of piperidine derivatives. Development of metal triflate- catalyzed diastereoselective nucleophilic substitution reactions of 2-methoxy- and 2- acyloxypiperidines. J. Org. Chem. 2001, 66, 809-823
109 O CF3SO3
O Ph Ph Ph
Oxonium ion 24'
The attack by the nucleophile 25 on the oxonium ion 24′ was favored from the less
hindered face and gave trans-substituted compounds as the major products. That approach helped
us to control the stereochemistry at the carbon C2′. That synthetic approach used would yield the
13-epi isomer of lucilactaene. LDA and LHMDS were used as bases during the preparation of
silyl enolate to maximize either E enolate or Z enolate in order to optimize the S diastereomer or
R diastereomer. The two trans diastereomers 26a and 26b were separated, but during the subsequent removal of the silyl protecting group with TBAF a mixture of two diastereomers
28a/28b was obtained regardless the single diastereomer used. The fluoride ion, being a strong base, deprotonated the acidic proton Hβ, and reprotonation during work-up gave two inseparable diastereomers 28 (Scheme 6-1) in a 45/55 ratio. The resulting alcohols were oxidized to the keto aldehydes 29a and 29b, using Dess-Martin periodinane.81
81 Martin , J. C.; Dess, D. B. A useful 12-I-5 triacetoxyperiodinane (the Dess-Martin periodinane) for the selective oxidation of primary or secondary alcohols and a variety of related 12-I-5 species, J. Am. Chem. Soc. 1991, 113, 7277-7287
110 Scheme 7.2 Removal of the silyl group
O OSi Bu4N O H O O H O O O TBAF OCH3 OCH3 OTr OTr
H3O
O O OH OH H H H H O O O O + OCH3 OCH3 OTr OTr
After oxidation, the resulting ketoaldehydes were coupled with the different tributylphosphonium salts 10 to yield two diastereomers, compounds 30a and 30b after separation using the previous reported condition.68 The same difficulty encountered in the reported conditions was also encountered, and a moderate yield (around 60%) was only achieved when a five fold excess of tributylphosphonium salt was used and the reaction temperature was maintained around -46 ºC.
111 2.2.5. Coupling fragment III and V
Scheme 8. Synthesis of tetraenes 30a and 30b
O O
O H H H O O R O 30a and 30b O O OCH3 OCH H 3 O O OTr Bu P a + 3 + Br- OCH3 O O OTr R 10a or 10b O 29a and 29b H H O O R
OCH3 a) t-BuOK, 18-crown-6, THF, -46 0C OTr 31a and 31b
82 The trityl group of diastereomer 30b was deprotected using BCl3 to give the secondary alcohols 32b. An attempt to form the intermediate lactone with DABCO or DBU in CH2Cl2
resulted in the decomposition of compound due to the acidic proton between the two carbonyls.
The ketone was then reduced using Luche reduction procedure to form a diol. The treatment of
the diol with base resulted in cleavage of the molecule in two (retro aldol condensation)
82 Jones, G. B.; Hynd, G.; Wright, J. M.; Sharma, A. On the selective deprotection of trityl ethers, J. Org. Chem. 2000, 65, 263-265
112 Scheme 9 Trityl deprotection
O O O O
O O H H BCl H H O O 3 O O N 31b 32b OCH3 S OCH3 S OTr OH
DBU or dabco
No lactonization, decomposition
O O O O
O HO H H H H O O O O N CeCl3 7H2O, NaBH4 N 32b 33b OCH3 S OCH3 S OH OH
Base
O O H O O O + OCH3 H OH N S
113 2.2.6. Suggested work: Enzymatic lactonization
The future goal of the project will be to try to form the lactone intermediate using lipase
from porcine pancreatic and if the lactonization occur, the lactone intermediate 33a and 33b will be opened with NH3 in methanol to yield the alcohol amide intermediates 34a and 34b which will then oxidized by Dess-Martin to afford lucilactaene and its analogues.
O O O OCH3 O O Lipase H H H H O O R O R O OCH3 O OH 32b
O OCH O OCH3 3 O O NH /MeOH H 3 H O O R O R O 33 34 O NH2 OH
O OCH 3 O OCH3 O O H H O O R Dess-Martin O R O 34a periodinane Lucilactaene NH2 NH OH and analogues OH
114 3. Conclusion
A synthetic approach to the total synthesis of lucilactaene was presented. The
lactonization step was not successful, so the goal to synthesize lucilactaene was not achieved.
4 Experimentation
4.1 General Experimental Conditions
All reactions were run under an atmosphere of dry nitrogen or argon. Solvent were dried by distillation from CaH2 for CH2Cl2 and from Na/benzophenone for THF. Reagents were purchased from Aldrich or Alfa Aesar. NMR spectra were obtained on a JEOL Eclipse NMR spectrometer or a Varian INOVA Spectromer 500 MHz and 400 MHz for protons and carbons respectively.
4.2 Diethyl di-iodomethylmethylmalonate (1).
Diethyl methylmalonate (44 g, 0.253 mol) added was droppewise to sodium hydride (60% in mineral oil; 10.2 g, 0.253 mol) in dry diethyl ether 300 ml over 1 h with vigorous stirring and the resulting thick mixture was refluxed for 2 h. Iodoform (100 g, 0.253 mol) was then added in one portion and the mixture was refluxed for 24 h under nitrogen. The mixture was then cooled to 0
ºC (ice-water bath) and 10% aqueous HCl (200 mL) was added. The resulting solution was stirred for 20 min. The organic layer was decanted, dried with MgSO4, and concentrated. The remaining residue was diluted with pentane (100 mL) and the precipitated iodoform was removed by filtration. The solvent was evaporated under reduced pressure. The dark pink residue was purified by flash chromatography (silica gel, hexane/ethyl acetate, 9/1) to yield compound 1
115 (70 g, 63%) as a pink pink oil. δH H NMR (500 MHz JEOL, CHCl3): δH 5.95 (1 H, s, CHI2), 4.33
(4 H, q, J = 6.1, OCH2), 1.92 (3 H, s, Me), 1.4 (6 H, t, J = 6.0, OCH2Me)
4.3 (E)-3-Iodo-2-methyl-2-propenoic-2-acid (2).
A solution of diethyl di-iodomethylmethylmalonate 1 (70 g, 0.16 mol) and KOH (27 g, 0.48 mol) was heated to reflux in EtOH-water (3:1, 300 mL) for 24h. After cooling to room temperature, the solvent was removed under reduced pressure and the residue was redissolved in 10% aqueous
K2CO3 (300 mL) and washed with CH2Cl2 (3 x 100 mL). The basic solution was acidified with
12M HCl, extracted CH2Cl2 (3 x 100mL), dried with MgSO4, and concentrated to a solid which was recrystallized from light petroleum to afford the acid 2 (25 g, 74%) as white needles, mp.51-
53 ºC. δH H-MNR (500 MHz JEOL, CHCl3): 10.82 (1 H, s, CO2H), 7.91 (1 H, d, J = 1, CHI),
1.98 (3 H, d, J = 1, Me)
4.4 (E)-3-Iodo-2-methylprop-2-en-1-ol (3).
A solution of (E)-3-iodo-2-methyl propenoic-2- acid (2, 25g, 118mmol) in dry 100 ml THF was cooled to 0 ºC (ice-water bath) and LiAlH4 (118 mL, 1.0 M solution, 118 mmol) was added dropwise over 1 h. After being stirred at room temperature for overnight, the reaction mixture was recooled to 0 ºC and the excess hydride was carefully quenched with saturated aqueous
Na2SO4. Diethyl ether (200 mL) was added and the mixture was poured into cold 2M H2SO4
(300 mL), then the organic layer was decanted off and the aqueous layer extracted with CH2Cl2
(2 x 100 mL). The combined organic solutions were concentrated and the resulting oil was
dissolved in CH2Cl2 (200 ml) and washed with 10% aqueous K2CO3 (100 mL). The basic
aqueous phase was extracted with CH2Cl2 (2 x 100 mL), and the combined organic solutions
116 were dried over MgSO4. Removal of the solvent under vacuum gave oil residue which was
purified through silica gel chromatography (hexane- ethyl acetate, 1/1) to afford compound 3
1 (16.3 g, 70%). δH H MNR (500 MHz, CHCl3): 6.26 (1 H, t, J = 1.4, CHI), 4.09 (2 H, s, CH2O),
2.71 (1 H, br s, OH), and 1.83 (3 H, d, J = 1.4, Me); δC 147.2 (C-2), 77.2 (C-3), 67.0 (C-1), 21.3
(Me)
4.5 (E)-3-Iodo-2-methylallyloxyl (tert-butyl)diphenylsilane) (4).
TBDPSCl (10 mL, 78 mmol) was added to a solution of (E)-3-iodo-2-methylprop-2-en-1-ol (3)
(14.00 g, 78 mmol) and imidazole (7.28 g, 214 mmol) in DMF (100 mL) at room temperature and the reaction mixture was stirred for 2 h at this temperature. A saturated aqueous NaHCO3
solution was added, the organic material was extracted with ether, and the combined organic
extracts were washed with water, dried over anhydrous MgSO4, and concentrated under reduced
pressure. The crude oil was purified by gel flash chromatography on silica (3 % ethyl acetate in
1 hexane) to afford 29.8 g (96%) of TBDPS ether 4 as a colorless oil: H NMR (500 MHz, CDCl3)
δ 1.06 (s, 9 H), 1.75 (s, 3H), 4.11 (s, 2H), 6.29 (s, 1H), 7.35-7.44 (m, 6H), 7.63-7.66 (m, 4H); 13C
NMR (500 MHz, CDCl3), δ 19.3, 21.2, 26.8, 67.6, 76.0, 127.8, 129.8, 133.1, 135.5, 146.4;
+ HRFABMS found m/z 435.0638 [M+1]; calcd for C20H24OISi (M - H) 435.0641.
4.6 General procedure for the condensation of bromoylides with aldehydes.
A solution of dry CH2Cl2 (100 mL) containing 1 equivalent of Ph3CHCO2Me was cooled at -20
°C. N-bromosuccinimide (1.1 equivavent) was added to the cooled and the solution was stirred for 20 min at -20 °C, and then 2.5 equivalents of K2CO3 were added followed by aldehyde (1 equivalent ). The stirred reaction mixture was then heated to reflux for 24 h. After filtration and
117 washing with 50 mL CH2Cl2, the solvent was evaporated under reduced pressure and 100 mL of
ether was added to precipitate most of the triphenylphosphine oxide. The product was purified
chromatography on silica gel using hexane/ethyl acetate as mobile phase to afford (Z) α-bromo-
αβ-unsaturated esters 6.
4.7 (Z)-Methyl-2-bromobut-2-enoate (6a).
Ph3CHCO2Me (15.2 g, 45.4 mmol), N-bromosuccinimide (8.9 g, 50 mmol), and K2CO3 (15.7 g,
113.6mmol) were reacted with ethanal (2 g, 45.4 mmol) in CH2Cl2 for 24 h according to the general procedure to give compound 6a (5 g, 61 %, Z product) after chromatography. 1H NMR
13 (500MHz, CDCl3); δ 1.88 (d, 3H, J = 6.8 Hz), 3.75 (s, 3H), 7.32 (q, 1H, J = 6.8 Hz); C NMR
(500MHz, CDCl3); d 10.76, 46.01, 110.05, 110.05, 134.54, 155.83
4.8 (Z)-Methyl 2-bromo-3-(2-methyl-thiazol-4-yl) prop-2-enoate (6b).
A solution of ethyl 2-methylthiazole-4-carboxylate (3 g, 17.5 mmol) in toluene (60 mL) was
cooled at -78 ºC under nitrogen and 1M solution of DIBAL in toluene (26.3 mL , 26.3 mmol)
was added dropwise over 30 min and the reaction mixture was stirred at that temperature for 2 h.
The excess of hydride was destroyed by adding methanol (3 mL), and the reaction was diluted
with diethyl ether (100 mL). Saturated potassium sodium tartrate (100 mL) was then added and
the mixture was stirred for 1 h at room temperature. The organic layer was decanted and the
aqueous layer was extracted with diethyl ether (2x200 mL). The combined organic solvent was
dried over Na2SO4 and the solvent was removed under reduced pressure to afford 2-
methylthiazole-4-carbaldehyde (2 g 90 %) which was used for the next step without further
purification.
118 Ph3CHCO2Me (5.26 g, 15.7 mmol), N-bromosuccinimide (3.1 g , 17.3 mmol), and K2CO3 (7 g,
39.3 mmol) were reacted with 2-methylthiazole-4-carbaldehyde (2 g, 15.7 mmol) in CH2Cl2 for
24 h according to the general procedure to yield compound 6b as the major product ( 2.3 g , 57
1 %, Z) after column chromatography (hexane/ethyl acetate:8/2). H NMR (500MHz, CDCl3); δ
2.73 (s, 3H), 3.89 (s, 3H), 8.33 (s, 1H), 8.89(s, 1H).
4.9 General procedure for the Stille coupling.
tert-Butyllithium (1.7 M in pentane, 2.5 equiv.) was added dropwise to a solution of TBDPS
ether 4 (1 equiv) in THF (50 mL) at -78 °C, and the reaction mixture was stirred for 10 min at
this temperature. A solution of trimethyltin chloride (1.0 M in THF, 2 equiv) was added
dropwise at -78 ºC and the stirring was continued for another 20 min at this temperature. A
saturated aqueous NH4Cl solution was added and the organic material was extracted with ethyl
acetate, the combined organic extracts were dried over anhydrous MgSO4, filtered and
concentrated under reduced pressure to yield vinylstannane (5) which was used without further
purification. Bromo 6 (1 equiv), Pd (PPh3)4 (10% mol.), CuCl (10 equiv) and LiCl (12 equiv) were added to a solution of crude 5 in DMSO/THF (1/1) (50 mL). The reaction mixture was degassed by three freeze-thaw cycles. After that, the flask was covered with aluminum foil and the reaction mixture was stirred and heated at 60 ºC for 48 h. The reaction mixture was then diluted with ether (50 mL) and 3% NH4OH (50 mL), and stirred for 30 min. The organic phase was separated and the aqueous phase was extracted with EtOAc (2 x 200 mL). The organic phase was dried over Na2SO4 and the solvent was concentrated under vacuum. The resulting residue was purified by flash chromatography (silica gel, 3-10% ethyl acetate in hexane) to give compound 7.
119 4.10 (2E, 3E)-Methyl 2-ethylidene-4-(tert-butyldiphenylsilyloxymethyl) pent-3-enoate (7a).
TBDPS ether compound 4 (10.7 g, 24.6 mmol) was treated tert-butyllithium (1.7 M in pentane,
36 mL, 61.3 mmol); then followed by trimethyltin chloride (1.M solution in THF, 50 mL, 50
mmol) and work up according to the general procedure described above. Compound 6a (2.2 g,
12.3 mmol), Pd(PPh3)4 (2.8 g, 2.42 mmol, 10% mol.), CuCl (12.2 g, 122.9 mmol, 10 equiv) and
LiCl (6.25 g, 147.5 mmol, 12 equiv) were added to a solution of the crude 5 in DMSO/THF(1/1)
(50 mL), and the reaction mixture was stirred and heated at 60 ºC for 48 h. The resulting product
was purified by flash chromatography (silica gel, 3-10% ethyl acetate in hexane) to give
1 compound 7a (2.7 g, 55%) as a colorless oil: H NMR (500 MHz, CDCl3), 1.08 (s, 9H), 1.48 (s,
3H), 1.73 (d, J = 7.2 Hz, 3H), 3.74 (s, 3H), 4.19 (s, 2H), 6.17 (brs, 1H), 6,93 (q, J = 7.2 Hz,1H),
13 7.34-7.46 (m, 6H), 7.69-7.74 (m, 4H). C NMR (500 MHz, CDCl3), δ 156.3, 15.6, 51.8, 67.8,
117.9, 130.0, 139.6, 140.5, 167.9; The NMR data agreed with the literature data.68 HRFABMS,
+ found m/z 409.2175 [M+1] ; calcd for C25H33O3Si, 409.2199.
4.11 (2E, 3E)-[Methyl2ethylidene]-4-hydroxymethylpent-3-enoate (8a).
TBAF (1.0 M in THF, 9.4 mL, 9.4 mmol) was added to a solution of diene 7a (2.7 g, 6.6 mmol) in THF (40 mL) at 0 ºC. The reaction mixture was allowed to warm slowly to room temperature and stirred for 5 h. Water was added, the organic material was extracted with ethyl acetate, and the combined organic extracts were washed with water, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The oily residue was purified by flash chromatography
(silica gel, 15-40% ethyl acetate in hexane) to afford 1.0 g (90%) of alcohol 8a as a colorless oil:
1 H NMR (500 MHz, CDCl3) 8 1.56 (s, 3H), 1.63 (br s, 1H), 1.74 (d, J = 7.3 Hz, 3H), 3.74 (s, 3H),
13 4.16 (s, 2H), 6.04 (s, 1H), 6.95 (q, J = 7.3 Hz, 1H); C NMR (500 JOEL MHz, CDCl3) δ 15.3,
120 15.6, 51.8, 67.8, 117.9, 130.0, 139.6, 140.5, 167.9; The NMR data agreed with the literature
68 1 + data. H RFABMS found [M+1] calcd for C9H14O3 171.1021, found 171.1019.
4.12 (2E, 3E)-Methyl 4-(hydroxymethyl)-2-(2-methylthiazol-4-yl)methylene) pent-3.enoate
(8b).
Compound 7b was prepared by a similar procedure to that described above. Compound 6b (2 g,
7.6 mmol), Pd(PPh3)4 (1.7 g, 1.5 mmol, 10 mol %), CuCl (7.5 g, 76.0 mmol, 10 equiv) and LiCl
(3.9 g, 91.2 mmol, 12 equiv) were added to a solution of crude 5 in DMSO/THF(1/1) (50 mL), and the reaction mixture was stirred and heated at 60 ºC for 48 h. The resulting product was purified by flash chromatography (silica gel, 5-10% ethyl acetate in hexane) to give a difficultly separable mixture of compounds 7b and 6c (4 g). The mixture (4 g) was treated with TBAF (1.0
Min THF, 12.7 mL, 12.7 mmol) at 0 ºC to room temperature for 5 h. After work-up, the resulting product was purified by flash chromatography (silica gel, 10-60% ethyl acetate in hexane) to
1 yield compound 8b as a light yellow solid (1.6 g, 83% two steps). H NMR (500 MHz, CDCl3), δ
1.47 (s, 3H), 2.64 (s, 3H), 3.73 (s, 3H), 4.12(s, 2H), 6.25 (s, 1H), 7.2 (s, 1H), 7.38 (s, 1H), 7.66 (s,
13 1H); C NMR (500 MHz, CDCl3), δ 15.74, 19.20, 52.34, 67.81, 118.93, 121.66, 128.74, 132.41,
+ 141.78, 151.19, 165.22, 168.30. HRFABMS [M+1] 254.0848 found, calcd for C12H16NO3S
254.0851.
4.13 (2E, 3E)-Methyl 2-ethylidene-4-(bromomethyl)pent-3-enoate (9a).
To a stirred solution of 8a (0.9 g, 5.3 mmol) in CH2Cl2 (40 mL) were added CBr4 (2.46 g, 7.42 mmol) and Ph3P (1.95 g, 7.42 mmol) at 0 ºC. The reaction mixture was stirred for 10 min at room temperature, then quenched with saturated aqueous NaHCO3 and extracted with ether (40
121 mL). The organic layer was washed with water and brine, dried over Na2SO4 and concentrated under reduced pressure to yield crude product. The residue was purified by silica gel column chromatography (0-3% ethyl acetate in hexane) to afford the bromide 9a (1.1g, 90%) as colorless
oil. 1H NMR (500 MHz, CDl3), δ 1.69 (d, J = 1.2 Hz, 3H), 1.75 (dd, J = 7.3 Hz, 3H), 3.74 (s,
13 3H), 4.07 (s, 2H), 6.17 (brs, 1H), 6.99 (q, J = 7.3 Hz); H CNMR (500 MHz, CDl3), δ 15.5,16.6,
39.8, 51.9, 123.4, 129.6, 137.2, 140.8, 167.3. The NMR data agreed with the literaturedata68
+ + HRFABMS [M] calcd for C9H13BrO2 M 232.0099, found 232.0093.
4.14 (2E, 3E)-Methyl 4-(bromomethyl)-((2-methylthiazol-4-yl)methylene)pent-3-enoate (9b).
Compound 8b (1.5 g, 5.9 mmol) was reacted with CBr4 (2.7g, 8.3 mmol) and Ph3P (2.2 g, 8.3 mmol) in CH2Cl2 (40 mL) according to the procedure described above. The residue was purified by silica gel column chromatography (15-50% ethyl acetate) to afford the bromide 9b (2.1 g,
1 84%) as alight dark yellow oil. H NMR (500MHz, CDCl3); d 1.57 (s, 3H), 2.66 (s, 3H), 3.73 (s,
13 3H), 4.08 (s, 2H), 6.34 (s, 1H), 7.43 (s, 1H), 7.71 (s, 1H). C NMR (500MHz, CDCl3); d 16.69,
19.12, 44.27, 52.28; 121.99, 124.53, 127.75, 133.39, 137.57, 150.61, 165.18, 167.53
+ HRFABMS ,m/z [M+1] found 316.0003, calcd for C12H14NO2S 316.0007.
4.15 Tributyl-[(2E,4E)-4-methoxycarbonyl-2-methylhexa-2,4-dienyl]phosphonium bromide
(10a).
Tributylphosphine (1.5 mL, 6.1 mmol) was added to a solution of the bromide 9a (1.1 g, 4.7 mmol) in acetonitrile (30 mL) and the reaction mixture was heated under reflux for 3h. The solvent was removed under reduced pressure and the resulting residue was purified by flash chromatography (silica gel , 0-5% methanol in dichloromethane) to yield 1.6 g (96%) of
122 1 phosphonium salt 10a as a colorless oil: H NMR (500 MHz, CDCl3) 8 0.99 (t, J = 7.0 Hz, 9 H),
1.50-1.68 (m, 12 H), 1.71-1.78 (m, 3H), 1.76 (s, 3H), 2.45-2.56 (m, 6H), 3.69 (d, J = 15.8 Hz,
2H), 3.74 (s, 3H), 6.06 (br d, J = 5.0 Hz, 1 H), 7.10 (q, J-- 7.1 Hz, 1 H). 13C NMR (500 MHz,
CDCI3) 8 13.4 (x 3), 15.8, 19.2 (d, J = 46.2 Hz) (x 3), 20.4, 23.9 (x 3), 24.1 (x 3), 29.3 (d, J =
44.6 Hz), 51.9, 126.8, 127.0, 129.0, 141.3, 166.7; HRFABMS calcd for C21H40O2P (M - Br) +
355.2766, found 355.2776. The NMR data agreed with the literature data.68
4.16 Tributyl-[(2E,4E)-4-methoxycarbonyl-2-methyl-5-(2-methylthiazol-4-yl)-penta-
2,4-dienyl]-phosphonium bromide (10b).
Compound 9b (2.0 g, 6.3 mmol) was reacted with Tributylphosphine (2.1mL, 8.85 mmol) according to procedure described above. The resulting product was purified by silica gel column chromatography (0-10% methanol in dichloromethane) to yield compound 10b (2.5 g, 90%) as a
1 light viscous oil. HNMR (500Hz, CDCl3); d 0.87 (t, 6H, J = 4.8 Hz), 1.45 (q, 9H, J= 7.3 Hz),
1.55 (m, 6H), 2.43 (dd, 2H, J = 3.7, 13.5 Hz), 3.72 (s, 3H), 6.36 (s, 1H), 7.59 (s, 1H), 7.65 (s, 1H)
13 + C NMR (500Hz, CDCl3); HRFABMS calcd for C24H41O2PS (M - Br) 453.2825, found
453.2822.
4.17 (E)-Ethyl 4-hydroxybut-2-enoate (12).
A solution of monoethyl fumarate (10 g, 69.0 mmol) under nitrogen was cooled at -10 ºC using an ice-salt bath. A 1M solution of borane-THF complex (70 ml, 70 mmol) was added dropwise over 1h. The reaction mixture was maintained at-10 ºC, then gradually allowed to warm to room temperature and stirred overnight. The reaction was then quenched by dropwise addition of methanol. The solvent was removed under reduced pressure to give oil residue. The product was
123 purified by silica gel flash chromatography (hexane- ethyl acetate, 1/1) to afford compound 12
(6.5g) as a colorless oil and was used in the next step.
4.18 (E)-Ethyl-4-(tert-butyldimethylsiloxy) buta-2-enoate (13).
TBDMSCl (5.8 g, 39 mmol) was added to a solution of 12 (7.00 g, 36 mmol) and imidazole
(7.28 g, 107 mmol) in DMF (100 mL) at room temperature, and the reaction mixture was stirred for 4 hour. A saturated aqueous NH4Cl solution was added to quench the reaction, followed by
Hexane (200 mL) was also added. The organic material was extracted with ether, and the combined organic extracts were washed with water, and dried over anhydrous MgSO4. The solvent was removed under reduced pressure to yield oil residue. Column chromatograpy of the residue on silica gel with hexane–ethyl acetate (9:1) gave compound (13), (6.3g, 70%) as a
1 colorless oil. HNMR ( 500 MHz, in CDCl3), δ 0.06 (s, 6H) , 0.92 (s, 9H), 1.32 (T, 3H, J = 7.4
Hz, 4.20 (q, 2H, J = 7.4 Hz), 4.35 (dd, 2H, J = 2.0, 3.2 Hz), 6.10 ( dt, 1H, J = 2.0, 15.3 Hz), 7.00
(dt, 1H, J 3.2, 15.3 Hz). 1H NMR data agreed the literature data.
4.19 (E)-4-(tert-Butyldimethylsiloxy)-2-buten-2-ol (14).
A solution of compound 13 (6 g, 46.0 mmol) in toluene (100 mL) was cooled at -78 ºC under nitrogen and 1M solution of DIBAL in toluene ( 106 mL, 106 mmol) was added dropwise over
40 min and the reaction mixture was stirred at that temperature for 2h. The excess of hydride was destroyed by adding methanol (3mL), followed by the addition of diethyl ether (100 mL).
Saturated potassium sodium tartrate (100 mL) was then added and the mixture was stirred for 1 h at room temperature. The organic layer was decanted and the aqueous layer was extracted (2 x
200 mL) with diethyl ether. The combined organic solvent was dried over Na2SO4 and evaporated to afford compound 14 in quantitative yield. TLC revealed that product was pure so it
124 13 was used for the next step without further purification. C NMR (500 MHz, CDCl3), d -5.35,
25.85, 62.76, 63.09, 129.09, 130.58.
4.20 (E)-4-(tert-Butyldimethylsiloxy)-2-butenal (15).
A solution of DMSO (7.4 mL, 104.3 mmol) in CH2Cl2 (150 mL) was cooled to -78 ºC under nitrogen and a 2 M solution of oxalyl chloride in CH2Cl2 (26 mL, 26 mmol) was added over 30 min. The reaction mixture was stirred for an additional 30 min, then a solution of alcohol 14
(6.5g, 32.1 mmol) in CH2Cl2 (10 mL) was slowly added over 30 min. The resulting mixture was
stirred further for 45 min at -78 ºC, then i-Pr2NEt (35.4 mL, 208 mmol) was added over 45 min.
The mixture was stirred further for 30 min at -78 ºC and then slowly warned to 0 ºC by removing
the cooling bath. The reaction was quenched with aqueous NaHSO4 solution (1.0 M, 200 mL)
and stirred vigorously. The layers were separated, and the aqueous phase was extracted (3 x
Et2O). The combined organic layers were concentrated (30 ºC water bath), diluted with ether
(400 mL), washed with aqueous NaHSO4 (3 x 300 mL), water (2 x 200 mL), saturated aqueous
NaHCO3 (1 x200 mL) and brine (1 x200 mL). The organic solvent was dried over Na2SO4 and
concentrated to give the corresponding aldehyde 15 in quantitative yield as pale yellow oil which was used in the next step without further purification.
4.21 (2E, 4E)- Ethyl 6-(ter-butyldimethylsilyoxy)-2-methylhexa-2,4-dienoate (16).
To a solution of commercial available Ph3P=CCO2Et(CH3) (15.1 g, 41.7 mmol) in CH2Cl2 (100 mL) was added aldehyde 15 (6.4 g, 32.1 mmol) in CH2Cl2 (20 mL). The reaction mixture was refluxed for 18 h. After cooling, the solvent was removed under reduced pressure and ether (100 mL) was added to precipitate phosphine oxide, and the solution was filtrated through Celite. The solvent was removed under reduced pressure to yield oily residue. The product was purified by
125 silica gel column chromatography with (10% ethylacetate in hexane) to give compound 16 (7.7 g,
1 85%) as light yellow oil. 1HNMR (500 MHz, in CDCl3), δ H NMR (500 MHz, CDCl3),δ 0.00 (s,
6 H), 0.84 (s, 9H), 1.21 (t, 3 H, J = 7.1 Hz), 1.85 (s, 3 H), 4.18 (q, 2H, J = 7.1 Hz,) 4.22 (dd, 2H,
J = 4.3, 2.2, 2H), 6.02 (t, 1 H, J = 4.4 Hz), 6.08 (t, 1H, J = 4.4 Hz), 6.54 (t, 1H, J = 1.8 Hz), 6.56
(dd, 1H, J = 3.4, 1.8 Hz), 6.59 (t, 1H, J = 1.8Hz); 13C NMR, δ -5.39, 12.45, 14.21, 18.26, 25,78,
60.38, 63.10, 124.37, 126.63, 137.40, 140.08, 168.34. HRFABMS, found m/z 284.1806 [M+1]+; calcd for C15H28O3Si, 284.1808.
4.22 6(2E, 4E)-6-(tert-butyldimethylsilyoxy)-N- methoxy-N, 2-dimethylhexa-2, 4-dienamide
(17).
N,O-dimethyl-hydroxylamine hydrochloride (10.3 g, 105.6 mmol) was suspended in THF (100 mL), and the solution was cooled to -20 °C under nitrogen. A solution of i-PrMgCl in THF
(105.6 mL, 2.0 M, 211.2 mmol) was added over 40 min while maintaining the temperature below
-20 °C. The slurried reaction mixture was stirred at that temperature for another 40 min.
Compound 16 (7.5g, 26.4 mmol) was then added, and the mixture was allowed to stir for 2 h between -20-10 °C. The reaction was quenched with saturated aqueous NH4Cl. The product was extracted using tert-butylmethyl ether and the organic solution was dried over Na2SO4 and concentrated. Chromatographic purification on silica gel afforded compound 17 (7.3 g, 92%) of
1 analytically pure product. HNMR (500 MHz, in CDCl3), δ 0.01 (s, 6 H), 0.85 (s, 9H), 1.9 (s, 3
H), 3.15 (s, 3 H), 3.56 (s, 3 H), 4.21 (dd, J = 4.3, 2.2, 2H), 5.86 (t, 1H, J = 4.6, 1 H), 5.89 (t, 1H,
J = 4.6), 6.39 (d, 1H, J = 11.2 Hz), 6.45 (t, 1H, J = 1.8 Hz), 6.47 (dd, 1H, J = 3.4, 1.8 Hz,), 6.50
(t, 1H, J = 1.8Hz); 13C NMR, δ -5.40, 14.13, 18.22, 25.75, 33.54, 60.87, 63.13, 123.97, 130.35,
+ 131.60, 136.90, 172.37. HRFABMS, [M+1] calcd for C15H30NO3Si, 300.1995, found 300.1991.
126 4.23 (4E, 6E)-Methyl 8-(tert-butyldimethylsilyloxy)-4-methyl-3 oxoocta-4, 6-dienoate (18).
A solution of LHMDS (70.2 mL, 1M, 70.2 mmol) in THF (100 mL) was cooled at -78 ºC under
nitrogen and methyl acetate (7 mL, 5.7 g, 77.2 mmol) was added dropwise over 10 min and the
reaction mixture was stirred at -78 ºC for 30 min. Compound 17 (7.0 g, 23.4 mmol) in THF (10
mL), was added dropwise. The reaction mixture was stirred at -78 ºC for 4 hours, then the
reaction solution was poured into 10% aqueous HCl (100 mL) and the product was extracted
with diethyl ether (2 x 200 mL). The organic phase was washed with water (2 x 100 mL) and brine (100 mL), dried over Na2SO4, and evaporated under reduced pressure to afford oily product.
The crude oil was purified by silica gel chromatography (10 ethyl acetate in hexane) to give
1 compound 18 (6.2 g, 85%). H NMR (500MHz, CDCl3); δ 0.07 (s, 6H), 0.91 (s, 9H), 1.88 (s,
3H), 3.70 (s, 3H), 3.73 (s, 2H), 4.31 (dd, 2H, J = 2.3, 3.6 Hz), 6.20 (t, 1H, J = 4.4 Hz), 6.23 (s,
1H, J = 4.3 Hz), 6.65 (t, 1H, J = 2.0 Hz), 6.68 (dd, 1H, J = 2.0, 3.9 Hz), 6.71 (t, 1H, J = 2.0 Hz),
13 7.01 (d, 1H, J = 11.2 Hz). C NMR (500 MHz, CDCl3), δ -5.3, 11.4, 18.3, 25.8, 44.7, 52.3, 63.0,
124.5, 134.9 139.8, 142.4, 168.6, 193.7. HRFABMS found m/z 313.1832[M+1]+; calcd for
C16H29O4Si, 313.1835.
4.24 (S).-2-(2,2 Dimethyl-5-oxo-1, 3-dioxolan-4-yl) acetic acid acid (19).
To a room temperature suspension of (S)-malic acid (15 g, 111.9 mmol) in 25 mL 2,2- dimethoxypropane was added PPTS (200 mg, 1.05 mmol), and the reaction mixture was stirred overnight. The reaction was partitioned between water and CH2Cl2, and the aqueous layer extracted twice more with CH2Cl2. The organic layers were combined, dried over Na2SO4, and
concentrated to give 11.7 g of 19 (57%) as awhite solid. Colorless needles (6.16 g, 50%) were
1 obtained by recrystailization from ethylacetate/hexane. H NMR (500MHz, CDCl3): 6 9.98 (br s,
127 13 1H), 4.70 (m, 1H), 2.91 (Ill, 2H), 1.58 (d, 6H, J = 15.3 Hz). C NMR (500MHz, CDCl3) 175.1,
172.1, 111.6, 70.6, 36.2, 27.0, 26.0. The NMR data agreed with the literature.72
4.25 (S)-5-(2-Hydroxyethyl)-2, 2-dimethyl-l, 3-dioxolan-4-one (20).
Acid 19 (6 g, 44.7 mmol) was dissolved in 50 mL THF under nitrogen and cooled to -78"C. A solution of BH3 •THF (38 mL, 1 M in THF, 38 mmol) was added over 1 hour, and the reaction mixture was allowed to warm to room temperature overnight. The solvent was removed reduced pressure and the crude mixture purified directly by flash chromatography (100% acetone) after the removal to yield 5.9 g (quantitative) as a light yellow oil. This unstable material was used in the next reaction without further purification.
4.26 (S).Dihydro-3-hydroxyfuran-2(3 H)-one (21).
Alcohol 20 (5.9 g, 36.8 mmol) and PPTS (200mg) were dissolved in 60 mL toluene and the mixture heated to 65 °C for 1 h. After cooling to room temperature, the solvent was removed under reduced pressure to yield oily product. The product was purified by silica gel column chromatography (silica gel 50-100%ethylacetate in hexane) to give compound 21 (2.7 g, 72%) as
1 a colorless oil . H NMR (500 MHz, CD3OD); δ 6 4.51 (dd, 1H, J = 8.4, 10.1 Hz), 4.42 (dd, 1H, J
= 2.0, 9.0 Hz), 4.20 (m, 1H), 3.70 (bs, 1H), 2.58 (m, 1H), 2.28 (m, 1H). 13C NMR (500 MHz,
78 CD3OD); δ 178.1, 67.4, 65.2, 30.8. The NMR data agreed with the literature.
4.27 (S)-Dihydro-3-(trityloxy) furan-2(3H)-one (22).
To a solution of triphenylmethyl chloride (10.6 g, 38.2 mmol, 1.5 equiv) and DBU (5.3 mL, 35.7 mmol, 1.4 equiv.) in CH2Cl2 (100 mL) was added compound 21 (2.6 g, 25.5 mmol) and the
128 reaction mixture was stirred at room temperature for 24 h. The reaction mixture was then washed
with water (2 x 50 mL) and dried over Na2SO4, and the solvent was removed under reduced pressure to yield a crude triphenylmethyl ether. The crude residue was purified by silica gel column chromatography (5-10% ethyl acetate in CH2Cl2) to give compound 22 as a white solid
1 (7.0 g, 80%). H NMR (500 MHz, CDCl3); d 0.77 (m, 1H), 1.48 (m, 1H), 3.69 (ddd, 2H, J = 1.8,
5.9, 9.1 Hz), 4.00 (ddd, 1H, J = 1.6, 9.2, 8.9), 4.26 (dd, 1H, J = 2.3, 8 Hz), 7.17 (brt, 9H, J = 7.1),
1 7.25 (brt, 9H, J = 7.1 Hz), 7.55 (brd, 6H, J = 7.3 Hz). H NMR (500 MHz, CDCl3); d 30.39,
64.51, 69.62, 88.32, 127.37, 128.10, 128.9, 143.72, 175.23. HRFABMS found m/z 345.1489
+ [M+1] ; calcd for C23H21O3 345.1491.
4.28 (3S)-2-Hydroxy-3-trityloxytetrahydrofuran-(23).
A solution of compound 22 (6.5 g, 18.9 mmol) in THF (100 mL) was cooled to -78 ºC under nitrogen and a solution of DIBAL in THF (37.8 mL, 1M, 37.8 mmol) was added dropwise over
30 min and the reaction mixture was stirred at that -78 ºC for 2h. The excess of hydride was destroyed by adding methanol (3 mL), followed by the addition of diethyl ether (100 mL).
Saturated potassium sodium tartrate (100 mL) was then added and the mixture was stirred for 1 h at room temperature. The organic layer was decanted and the aqueous layer was extracted with diethyl ether (2x200 mL). The combined organic extracts were dried over Na2SO4, the solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography (10-40% ethyl acetate in hexane) to afford compound 23 (6 g, 92%) as a semi-
1 13 solid. H NMR many overlapping peaks. C NMR (500 MHz, CDCl3); d 29.27, 31.27, 64.12,
67.02, 74.61, 78.97, 87.56, 88.16, 127.13, 127.44, 127.86, 128.08, 128.49, 128.82, 143.88,
+ 144.35, HRFABMS found m/z 347.1645 [M+1] ; calcd for C23H23O3 347.1647.
129 4.29 (3S)-2-Acetoxy-3-trityloxytetrahydrofuran (24).
Compound 23 (5.5 g, 15.9 mmol) was dissolved in pyridine and treated with acetic anhydride (X
mL, 95.4 mmol) and the reaction mixture was stirred overnight. The mixture was then poured
into ice water (100 mL), and the product was extracted with diethyl ether (3 x 300 mL). The
organic phase was washed with citric acid (2 x 100 mL, 20%), saturated sodium bicarbonate (2 x
100 mL) and finally brine solution (100 mL). The solvent was removed under reduced pressure
and the product was purified by silica gel column chromatography (10-30% ethylacetate in
hexane) to yield compound 24 as a single diastereomer, (5.5 g, 89.1%) as a white solid. 1H NMR
(500 MHz, CDCl3); δ 1.65 (m, 1H), 1.88 (m, 1H), 3.80 (ddd, 2H, J = 5.5, 8.2, 5.7 Hz), 4.04 (dd,
13 1H, J = 7.55, 8 Hz), 4.15 (dd, 1H, J = 2.5, 6.1 Hz). C NMR (500 MHz, CDCl3) δ 21.0, 31.5,
68.1, 78.1, 87.9, 100.9, 127.3, 127.91, 128.7, 144.1, 169.3. HRFABMS found m/z 389.1750
+ [M+1] ; calcd for C25H25O4 389.1753.
4.30 (4E,6E)-Methyl-2-(3S)-tetrahydro-3-(trityloxyl)-furan-2-yl)-8-tert-butyl-
dimethylsilyloxy-4-methyl-4-oxoocta-4, 6-dienoate (26, 27).
A solution of LHMDS (7.5 mL, 1M, 7.5 mmol) in THF (100 mL) was cooled at -78 ºC under
nitrogen and compound 18 (2.4 g, 7.7 mmol) in THF (10 mL) was added dropwise over 10 min
and the reaction mixture was stirred at that temperature for an additional 30 min. The reaction
was then treated with TMSCl (6 mL, 55.8 mmol) over 20 min and the reaction was stirred for an
additional 1 h. The solvent was then removed under vacuum and dried CH2Cl2 (20 mL) was added. The resulting solution was filtered under nitrogen to yield a solution of silyl enolate 25.
Compound 24 (1.5 g, 3.8 mmol) was added to the solution. The resulting solution was then added to dried CH2Cl2 (60 mL) containing Sc(OTf)3 (0.38 g , 20 mol %) cooled at 0 ºC. The
130 reaction mixture was stirred at 0 ºC for 4 h, monitoring with TLC. After 4 h, a saturated solution
of NaHCO3 was added and the mixture was stirred for 5 min. The organic phase was decanted
and the aqueous phase was extracted with CH2Cl2 (2 x 100 mL). The combined CH2Cl2 extract
was washed with water (200 mL), brine solution (200 mL), dried over Na2SO4, and evaporated under reduced pressure yield viscous oil residue. The crude product was purified by silica gel column chromatography to yield 4 diastereomers, and trans products as majors 26a/b (80%) and the cis products 27a/b as minor products, 20% of the mixture. The overall yield was 86%. 13C
NMR d -5.22, -5.24, 11.72, 11.77, 14.20, 14.26, 18.45, 18.49, 22.74, 25.94, 25.97, 31.67, 32.81,
52.48, 52.51, 55.97, 56.01, 63.22, 63.27, 67.63, 67.65, 77.42, 77.54, 84.07, 84.13, 87.47, 87.52,
124.77, 124.80, 127.20, 127.28, 127.80, 127.96, 128.21, 128.97, 129.07, 135.56, 135.61, 139.08,
139.21, 142.36, 144.45, 144.56, 144.61, 168.45, 168.52, 193.28, 193.32. HRFABMS found m/z
+ 641.3288 [M+1] ; calcd for C39H49O6Si 641.3298.
4.31 (4E,6E)-Methyl2-((3S)-tetrahydro-3-(trityloxyl)furan-2-yl)-8-hydroxy-4-methyl-
-3-oxoocta-4, 6-dienoate (28).
TBAF (1.0 M in THF, 3.2 mL, 3.2 mmol) was added to a solution of diene mixture 26a/b (0.9 g,
1.4 mmol) in THF (40 mL) at 0 ºC. The reaction mixture was allowed to warm slowly to room temperature and stirred for 5 h. Water was added, the organic material was extracted with ethyl acetate, and the combined organic extracts were washed with water, dried over anhydrous
Na2SO4, and concentrated under reduced pressure after filtration. The oil residue was purified by flash chromatography (silica gel, 15-40% ethyl acetate in hexane) yield alcohol 28a and 28b as a mixture of isomers.
131 13 C NMR (500 MHz, CDCl3), δ 11.78, 11.93, 32.72, 32.77, 52.43, 52.49, 55.79, 56.01, 67.41,
67.57, 77.26, 127.1, 127.87, 128.94, 138.49, 138.51, 141.25, 141.28, 144.50, 144.52, 167.89,
+ 168.32, 193.25, 193.68. HRFABMS found m/z 527.2428 [M+1] ; calcd for C33H35O6, 527.2434.
4.32 General Coupling Procedure of Fragments II and III.
Dess-Martin periodinane (1g, 2.42 mmol) was added to a solution of 28a and 28b (0.850g, 1.62 mmol) in CH2Cl2 and the reaction mixture was stirred for at room temperature. A solution of
Na2S2O3 (1 M, 20 mL) and a saturated aqueous NaHCO3 solution was added. The mixture solution was extracted with diethyl ether (2 x100 mL). The organic solution was washed water and brine dried over Na2SO4, filtered and evaporated under pressure to a mixture of aldehyde
29a and 29b (0.74g , 87%). The resulting aldehyde was used without further purification.
Potassium tert-butoxide (1.0 M in THF, 1 equiv.) was added to a solution of the phosphonium salt 10 (5 equiv) and 18-crown-6 (5 equiv.) in THF (1 mL) at -78 °C. The reaction mixture was allowed to warm to -46 ºC and stirred for 15 min. A solution of aldehyde (1 equiv.) in THF (1 mL) was added to the reaction mixture at -46 ºC and the stirring was continued for an- other 10 min at this temperature. A saturated aqueous NaHCO3 solution was added, the organic material was extracted with ethyl acetate, and the combined organic extracts were washed with brine, dried over anhydrous NaSO4, and concentrated under reduced pressure. The crude product was purified by PTLC (25% ethyl acetate in hexane) afforded compounds 30.
4.33 2-Ethylidene-4, 10-dimethyl-11-oxo-12-(3-trityloxy-tetrahydro-furan-2-yl)-trideca-3, 5,
7, 9-tetraenedioic acid dimethyl esters (30a) and (31a).
132 Compound 29a (200 mg, 0.38mmol) reacted with phosphonium salt 10a (0.82g, 1.9 mmol) in the presence of 18-crown-6 (0.5 g, 1.9 mmol) and Potassium tert-butoxide 1.0 M in THF (1.8 mL,
1.8 mmol) according to procedure described above. The resulting residue was purified by normal phase PTLC plate by developing three time (30% ethylacetate in hexane) to yield compounds
30a (75 mg) impure (29.8%) and 31a (80 mg, 31%) , 96% as yellow amorphous substance; 1H
NMR (500 MHz, CDCl3) (31a), δ 1.07 (m, 1H), 1.30 (dd, 1H, J =7.5, 15.1 Hz), 1.38 (m, 1H),
1.53 (dd, 1H, J = 7.5, 15.1 Hz), 1.67 (s, 3H), 1.68 (d, 3H, J = 7.3 Hz), 1.69 (s, 3H), 3.58 (s, 3H),
3.67 (s, 3H), 3.73 (ddd, 1H, J = 2.7, 8.2, 2.7 Hz), 3.81 (ddd, 1H, J = 1.8, 6.4, 1. 1.8 Hz), 4.72 (dd,
1H, J = 2.3, 4.6Hz), 6.14 (s, 1H), 6.37 (dd, 1H, J = 4.5, 10.5 Hz), 6.48 (dd, 1H, J = 11.0, 14.7),
6.51(d, 1H, J =15.1 Hz), 6.54 (dd, 1H, J = 11.5, 14.7), 6.81 (d, 1H, J = 11.7), 6.93 (q, 1H, J =
13 7.3). C NMR (500 MHz, CDCl3), δ 11.79, 14.25, 15.9, 32.67, 51.90, 52.38, 56.00, 67.51, 84.00,
87.35, 127.08, 127.51, 127.8, 128.11, 128.34, 128.96, 130.32, 135.49, 137.97, 139.67, 140.40,
141.18, 141.31, 144.50, 167.44, 168.38, 192.64. HRFABMS found m/z 661.3158 [M+1]+; calcd for C42H45O7.
4.34 4,10-Dimethyl-2-(2-methyl-thiazol-4-ylmethylene)-11-oxo-12-(3-trityloxy-tetra- hydro-furan-2-yl)-trideca-3, 5, 7, 9-tetraenedioic acid dimethyl ester (30b) and (31b).
Compound 29b (250 mg, 0.47 mmol) reacted with phosphonium salt 10b (1.23 g, 2.4 mmol) in the presence of 18-crown-6 (mg, X mmol) and potassium tert-butoxide 1.0 M in THF (2.3 mL,
2.3 mmol) according to procedure described above. The resulting residue was purified by normal phase PTLC plate (10% ethylacetate in hexane) to yield by developing three time to afford 30b
1 (90 mg, impure), 31b (100 mg, 28%) and 97 % pure. H NMR (500 MHz, CDCl3),(31b) δ 1.07
(m, 1H), 1.37 (dd, 1H, J =7.5, 15.1 Hz), 1.44 (m, 1H), 1.59 (dd, 1H, J = 7.5, 15.1 Hz), 1.73 (s,
133 3H), 1.76 (d, 3H, J = 7.3 Hz), 2.70 (s, 3H), 3.64 (s, 3H), 3.81 (s, 3H), 3.73 (ddd, 1H, J = 2.7, 8.2,
2.7 Hz), 3.81 (ddd, 1H, J = 1.8, 6.4, 1. 1.8 Hz), 4.72 (dd, 1H, J = 2.3, 4.6Hz), 6.14 (s, 1H), 6.37
(dd, 1H, J = 4.5, 10.5 Hz), 6.48 (dd, 1H, J = 11.0, 14.7), 6.51(d, 1H, J =15.1 Hz), 6.90(dd, 1H, J
13 = 11.5, 14.7), 7.70 (1H). C NMR (500 MHz, CDCl3) δ 11.72, 14.5, 19.13, 32.60, 52.24, 52.31,
55.95, 67.43, 77.33, 83.94, 87.29, 121.92, 127.04, 127.04, 127.80, 128.33, 128.33, 128.42,
128.42, 128.79, 128.9, 132.53, 135.56, 139.59, 141.03, 141.20, 144.43, 151.08, 165.97, 167.93,
+ 168.33, 192.59. HRFABMS found m/z 744.2991 [M+1] ; calcd for C45H46NO7S 744.2995.
4.35 General Procedure for Deprotection of the Trityl Group with BCl3.
To a solution of compound 30 (1 equiv.) in CH2Cl2 at -30 ºC was adde BCl3 (1.1 equiv., 1.0 M in
CH2Cl2) dropwise via syringe. The reaction mixture was stirred at that temperature for 30 min, quenched with MeOH anhydrous. The reaction solution was poured onto saturated solution of
NaHCO3 (30 mL), stirred for 5 min and extracted with CH2Cl2 (3 x 40 mL). The organic phase was washed with brine (2 x 50 mL), dried over Na2SO4 and evaporated under reduced pressure to give compound 32 after purification on a normal phase PTLC plate
4.36 12-(3-Hydroxy-tetrahydro-furan-2-yl)-4,10-dimethyl-2-(2-methyl-thiazol-
4-yl-methylene)-11-oxo-trideca-3, 5, 7, 9-tetraenedioic acid dimethyl ester 33b.
Compound 30b (90 mg, 0.12 mmol) reacted with BCl3 in CH2Cl2 (1.1 mL, 1.1 mmol) according to procedure described above. The resulting residue was purified by normal phase
PTLC plate (10% ethylacetate in hexane) to yield compound 32b (45 mg, 75%) as yellow
13 amorphous substance; C NMR (500 MHz, CDCl3) δ 11.69, 14.58, 19.17, 34.39, 52.30, 52.78,
57.33, 67.30, 75.12, 86.25, 121.96, 128.49, 128.72, 133.32, 139.19, 141.73, 141.81, 142.40,
134 151.06, 165.33, 167.94, 168.92, 195.04. HRFABMS found m/z 502.1889 [M+1]+; calcd for
C26H31NO7S 502.1899.
135
13C NMR of compound 2.3
180 160 140 120 100 80 60 40 20 PPM
136 1H NMR of Compound 3.1
9 8 7 6 5 4 3 2 1 PPM
137 13 C NMR of Compound 3.1 (C5H5N)
200 150 100 50 PPM
138 13 C NMR of Compound 3.1 (CDCl3)
200 150 100 50 0PPM
139 COSY spectrum of Compound 3.1 (CDCl3)
1 2 3 1
n o i s n e 4 m i D
t c e r i d n 5 I
M P P 6 7 8
8 7 6 5 4 3 2 1 PPM Direct Dimension
140 HMBC of compound 3.1
0 0 5 1
n o i s n e m i 0 D 0
t 1 c e r i d n I
M P P 0 5 1 0 0 2
8 7 6 5 4 3 2 1 PPM Direct Dimension
141 1H NMR of compound 3.2
9 8 7 6 5 4 3 2 1 PPM
142 13C NMR of compound 3.2
200 150 100 50 PPM
143 COSY spectrum of compound 3.2 0 2 1
n o i s n e m i D 4 t c e r i d n I
M P P 6 8
9 8 7 6 5 4 3 2 1 PPM Direct Dimension
144 1HNMR of compound 3.3
9 8 7 6 5 4 3 2 1 PPM
145 13C NMR of compound 3.3
180 160 140 120 100 80 60 40 20 PPM
146 1H NMR of compound 3.7
9 8 7 6 5 4 3 2 1 PPM
147 13C NMR of compound 3.7
180 160 140 120 100 80 60 40 20 PPM
148 1H NMR of compound 4.3
8 6 4 2 PPM
149 13C NMR of compound 4.3
180 160 140 120 100 80 60 40 20 PPM
150 COSY spectrum of 4.3 1 2 3 1
n o i s n e 4 m i D
t c e r i d n I 5
M P P 6 7 8
7 6 5 4 3 2 1 PPM Direct Dimension
151 HMBC spectrum of 4.3 0 0 5 1
n o i s n e m i D
t c e 0 r i 0 d 1 n I
M P P 0 5 1
9 8 7 6 5 4 3 2 1 PPM Direct Dimension
152 1H NMR of compound 5.6
6 5 4 3 2 1 PPM
153 13C NMR of compound 5.6
200 150 100 50 PPM
154 1H NMR of compound 5.7
7 6 5 4 3 2 1 PPM
155 13C NMR of compound 5.7
200 150 100 50 PPM
156 1H NMR of compound 5.2
7 6 5 4 3 2 1 PPM
157 13C NMR of compound 5.2
200 150 100 50 PPM
158 1H NMR of Compound 6.1
10 8 6 4 2 PPM
159 13C NMR of Compound 6.1
200 150 100 50 PPM
160 COSY spectrum of compound 6.1 2 - 0 2 1
n o i s 4 n e m i D
t c e r i d 6 n I
M P P 8 0 1
10 8 6 4 2 PPM Direct Dimension
161 HMBC spectrum 6.1 0 0 5 1
n o i s n e m i D
0 t 0 c 1 e r i d n I
M P P 0 5 1 0 0 2
10 8 6 4 2 PPM Direct Dimension
162 1H NMR of compound 6.2
10 8 6 4 2 PPM
163 13C NMR of Compound 6.2
200 150 100 50 PPM
164 COSY spectrum of 6.2 0 2 1 4
n o i s n e m i D
t c e 6 r i d n I
M P P 8 0 1
10 8 6 4 2 PPM Direct Dimension
165 HMBC spectrum of 6.2 0 0 5 1
n o i s n e 0 m i 0 1 D
t c e r i d n I
M P P 0 5 1 0 0 2
10 8 6 4 2 PPM Direct Dimension
166
1H NMR of Compound 6.3
8 7 6 5 4 3 2 1 PPM
167 13C NMR of Compound 6.3
180 160 140 120 100 80 60 40 20 PPM
168 COSY spectrum of compound 6.3 1 - 0 1 1
n 2 o i s n e m i D
t 3 c e r i d n I
M P 4 P 5 6
6 5 4 3 2 1 PPM Direct Dimension
169 HMBC of compound 6.3 0 0 5 1
n o i s n e m i D
t c 0 e r 0 i 1 d n I
M P P 0 5 1
6 5 4 3 2 1 PPM Direct Dimension
170 1H NMR of compound 6.5
10 8 6 4 2 PPM
171 1H NMR of compound 6.5
200 150 100 50 PPM
172 HMBC spectrum of compound 6.5 0 0 5 1
n o i s n e 0 m 0 i 1 D
t c e r i d n I
M P P 0 5 1 0 0 2
10 8 6 4 2 PPM Direct Dimension
173 1H NMR Compound 6.6
7 6 5 4 3 2 1 PPM
174 13C NMR Compound 6.6
180 160 140 120 100 80 60 40 20 PPM
175 O O Si O
O Fragment IV (18)
7 6 5 4 3 2 1 0PPM
176
O O Si O
O Fragment IV (18)
200 150 100 50 0 PPM
177 O O O
O
Fragment V (24)
8 7 6 5 4 3 2 1 PPM
178
O O O
O
Fragment V (24)
180 160 140 120 100 80 60 40 20 PPM
179
O O O H O O O O 30a
7 6 5 4 3 2 1
180 O O O H O O O O 30a
200 150 100 50 PPM
181 O O O H O O N O O S 31a
8 7 6 5 4 3 2 1 PPM
182 O O O H O O N O O S 31a
180 160 140 120 100 80 60 40 20 PPM
183
O O O H O O N O OH 32a S
8 7 6 5 4 3 2 PPM
184
O O O H O O N O OH 32a S
200 150 100 50 PPM
185 Vita
Eba Adou was born in Côte d’Ivoire. He attended “Université Nationale d’Abidjan” in Côte d’Ivoire where he earned a “C.E.S” in organic chemistry in 1989. He came to the U.S.A in June
1990 and commenced studies at The University of Texas at Austin in fall 1991, where he obtained a B.A. in chemistry in May, 1993. He entered Southwest Texas State University, San
Marcos, Texas in fall 1994, where he obtained a Master of Science in chemistry in May, 1997.
He came to Virginia Polytechnic Institute and State University in January 2000 and joined the natural products group of Professor David G. I. Kingston where he earned a Ph.D. in chemistry in fall 2005. He met his beloved wife, Dr. Nan Chi Wan, in 2000, and they were married on
March 19, 2005.
186