APPLICATIONS OF MODERN NMR METHODS TO STRUCTURAL STUDIES OF NATURAL AND

SYNTHETIC ORGANIC COMPOUNDS

Suzanne Monk B issada

A Thesis submitted in confomity with the requirements for the degree of Master of Science Graduate Department of Chemistry

University of Toronto

Q Siislanne M. Bissada, 1999.

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Abstract

The study of natural and synthetic organic compounds has been investigated.

The use of one-dimensional and two-dimensional NMR methods as a method for rapidly

assigning structures was demonstrated. Structurai elucidation was perfomed on

unknown side-products in synthetic sequences to conclusively determine what reactions

had taken place. Steps toward a biomimetic synthesis of the alkaloid, dihydrocadambine,

were carried out. The isolation of the nahu;il product precursor to this alkaloid,

secologanin, was first performed. in the course of isolating secologanin, two other

natural products, loganin and sweroside were also isolated and structurally elucidated.

The intermediates in the atternpted synthesis of dihydrocadambine were also examined.

Finally, NMR methods were applied to characterization of diterpene and sesquiterpene

naturai products isolated in the Caribbean. Eleven of the natural products investigated

were determined to be novel.

Ac knowledeemen ts

1 wish to express my sincere gratitude to my supenisors, Professor Stewart

McLean and Professor William F. Reynolds. The opporhmity to work with two such

experienced, knowledgeable. worldly and genuine people will always be appreciated. 1

am thankful for the guidance and patience that was readily provided to me.

The expenence of working with the Caribbean affiliates from the University of

West Indies and the Mexican affiliates at Universidad Nacional Autonoma de México

was truly a pleasure. Working in such an international comrnunity provided more than

just a scientific learning experience

1 would like to thank Dr. Timothy Burrows for al1 the extra help he gave me on

instrumentation in the NMR facilities at the University of Toronto. Thanks are also due

to Ms. Nura Hagi-Nur for her contribution in isolating the natural products fiom the

honeysuc kle plant.

1 cannot forget the people that have been a constant support throughout my life. 1

can never be thankful enough to al1 my parents, Faiza Fahim Bissada, Mo& Giryagos

Bissada, Samir Magar, Feria1 Magar and Sarnia Bissada, and my brothers, Freddy Farid

Tengberg, Yousry Bissada and Ramses Bissada for their undying support and patience.

For my mother

Faiza Fahim Bissada

iii

Table of Contents --

Abstract Acknowledgement Dedica tion Table of Contents List of Ta b1es List of Abbreviations

Chapter 1 - Description of NMR Methods

1 . 1 Introduction

1.2. 2D NMR Expenments

1.3. Chemical Shifts

1.4. Multiplicity and Coupling

1 S. Instrumental Experimental

Chapter 2- Structural Elucidation of a Synthetic Intermediate

2.1. Background

2.2. NMR Data for Structure 3

2.3. Structural Determination of 3

2.4. Interpretation of Data

Chapter 3 - Synthetic Plan

3.1. Previous Synthesis of 3a and 3P- dihydrocadarnbine

3.2. Synthetic Plan

Page i ii iii iv vi vii

Chapter 4 - Isolates from Lonicera tartarica

4.1 . Iridoids of Lonicera tartarica

4.2. General Experimental

4.3. Isolation of Secologanin fiom Lonicera tartarica

4+4. Products Isolated fiom Extract

4.5. Forming dimethyl acetal O f 9

Chapter 5 - Steps toward Synthesis of Dihydrocadambine

5.1. mCPBA oxidation of 8

5.2. Dihydroxylation of STAMA

5.3. Formation of Key Epoxide

5.4. Pictet-Spengler Reaction

5 S. Deprotection of Dimethyl Acetal

5.6. Coupling of Epoxide 28 to tryptarnine

5.7. Discussion of Results

Chapter 6 - NMR Analysis of Natural Products

6.1. Terpenes

6.2. Determination of Structures of Two Sesquiterpenes fiom Capraria

6.3. Elucidation of Unknown Caesalpinia bonduc Root Extracts

6.4. Structural Elucidation of Awiellamines A and B

References

List of Tables --

Table

2-1

4-1

4-2

5- 1

5-2

5-3

5-4

5-5

6-1

6-2

6-3

6-4

6-5

6-6

6-7

Titfe

500 MHz NMR data for 3

400 MHz NMR data for 22

500 MHz NMR data for 21

400 MHz NMR data for glycol 24

500 MHz NMR data for mono-epoxide 26

500 MHz I3c. ' H and HMBC data for 18a

500 MHz "c. 'H, COSY and HMBC for 27

500 MHz 13c, 'H md HMBC data for 29

500 MHz 13c. 'H and HMBC data for 30

500 MHz 13c, 'H and HMBC data for 31

500 MHz 'H and "C data for 32 and 33

500 MHz 'H and l3c data for 34 and 35

500 MHz 'H and "C data for 36 and 37

500 MHz 'H and I3c data for 38 and 39

500 MHz 'H and 13c data for 40 and 41

Page

12

27

3 1

37

39

43

46

52

58

60

64

66

69

List of Abbreviations --

COSY: Correlation Spectroscopy

EtOAc: ethyl acetate

HMBC: ' ~de t ec t ed multiple-bond heteronuclear multiple-quantum coherence)

HMQC: '~Ddetected heteronuclear multiple-quantum coherence

HRMS : high-resolution mass spectrometry

HSQC: 'H-detected heteronuclear single-quantum coherence

mCPB A: me tu-c hloro peroxy benzoic acid

n.0.e.: nuclear Overhauser effect or enhancement

NMO : N-methy lmo rpholine Nsxide

NMR: Nuclear Magnetic Resonance

NOESY: homonuclear Overhauser effect spectroscopy

pTSA: para-toluene sulfonic acid

ROESY: rotating frame NOESY

STA: secologanin tetraacetate

STAMA: secologanin tetraacetate dimethyl acetal

TBAF: tetnibutylammonium fluoride

TLC: Thin Layer C hromatography

TMSOTfi Trimethyl silyl tnfluoro methane sulfonate

Ts : p-toluenesulfony 1

vii

Chapter 1. Descriplon of NMR Methods

1.1. Introduction

Nuclear magnetic resonance (NMR) spectroscopic data provides chemists with

information that is indispensable for unraveling the structure of organic compounds. The

interface of synthetic chemistry and NMR spectroscopy is becoming more closely

entwined as both develop. The utility of NMR to elucidate the structure of both expected

and unexpected products is of vital importance. Previously, when a synthetic sequence

was desired, characteristics for the desired product were usually the only ones looked for.

A method for rapidly assigning the structures of synthetic intermediates would provide a

secure way of ensuring that the proposed synthetic route was, in fact, being followed.

Identifjing the structure and charactenstics of an unknown side-product can result in

interesting chemistry, and modem methods of NMR spectroscopy in combination with

other instrumentation techniques, such as mass spectrometry and infrared spectroscopy,

are ideal for this purpose. The use of NMR techniques by means of one-dimensional and

two-dimensional nuclear magnetic resonance (2D NMR) for structure elucidation of both

naturai products and synthetic products were examined in the course of this thesis

researc h.

1.2. 2D NMR Exiieriments 1 2 3 4

Multi-dimensional NMR experiments have revolutionized the use of NMR

spectroscopy for structure elucidation of al1 molecules varying fiom small molecules to

large complex proteins. 2D NMR experiments are designed to generate different kinds of

fiequency information dong the two axes. The preparation time is a relaxation delay,

followed by one or more pulses to start the experiment.

The two distinct mechanisms for magnetization transfer are scalar coupling and

cross-relaxation. Scalar coupling yields information on the proximity of spins through

the covalent framework of the molecule and the latter provides information on spatial

proximity and molecular motion. In COSY (correlated spectroscopy), the tl period is

initiated with a 90" 'H pulse. Magnetization transfer arnong the transitions of a coupled

system is mediated by a second 90" 'H pulse before the t2 period. As a result of net

magnetization transfer, cross-peaks appear between al1 transitions belonging to the same

coupled spin system. Heteronuclear 2-D experiments cm also be perfonned for exarnple

when 'H magnetization can be transferred to a 13c nuclei by means of a 90" 'H pulse

followed by a delay and a 90" 13c puise. By employing proper delays in the preparation

period, HMQC ('H-detected heteronuclear multiple-quantum coherence) or HSQC ('H-

detected heteronuclear single quantum coherence) provide correlation for directly bonded

'H and I3c pairs of nuclei. HSQC differs fiom HMQC in that 'H multiplet structure

5 appears alongfr in the latter sequence but not in the former. HSQC was found, in our

lab, to provide better 13c resolution and sensitivit$ and consequently was used in place

of HMQC throughout these investigations. Similarly, W C (I~~detected multiple-bond

heteronuclear multiple-quantum coherence) provides 'H-'~c correlation via long-range

coupling. In both HSQC and HMBC experiments, another 90" "C pulse prior to the tz

period transfers the magnetization back to the 'H nuclei for detection.

Overhauser effect spectroscopy is based on cross-relaxation which is an

interaction in which the spins exchange magnetization by fluctuating through-space

dipole-dipole couplings. As a result of the distance dependence of the dipolar interaction,

cross-peaks in the 2D spectra may indicate spatially proximate partners. NOESY

(homonuclear Overhauser effect spectroscopy) has a pulse sequence similar to that of

COSY, but with an extra delay and a 90" 'H pulse before the t2 penod. During this

delay, nOe (nuclear Overhauser effect or enhancement) develops dong the z-axis. The

nOe causes the change in intensity (increase or decrease) during decoupling experiments.

The nuclear Overhauser measurements can also be performed in the rotating fiame and

under these conditions nOe factors are always positive. ROESY (rotating fiame

NOESY) experiments are used for molecules tumbling at a rate where nOe crosses over

from positive to negative. ROESY experiments are particularly important for

intermediate molecular weight molecules (ca. 500- 1 500).

The real usefûlness of multi-dimensional NMR in general is not in the

information provided by a single expenment, but rather in the synergy provided by

canying out several different experiments on the sarne molecule. Combining al1 these

experiments together, aid in identifjing each nucleus in a molecule, and their relative and

spatial positioning. Thus the experiments can be used either for complete NMR spectral

assignments of known molecules or mapping the structure of unknowns. A schematic

illustration for the various 2D NMR experiments w d for structural elucidation is shown .

in Figure 1-1.

1. COSY

2. HSQC

3. HMBC

Figure 1-1 - Schematic ülustration of the procedures used for structural assignment. 1 .) COSY assigns sequences of coupled protons 2.) HSQC assigns carbons for the same sequences. 3.) HMBC assigns non-protonated carbons and ties together molecular fragments separated by non-protonated carbons or heteroatoms.

1.3. Chernical Shifts 'g8?

Chernical shifts reflect the extent to which the basic Larmor fiequency of a

nucleus is altered by its electronic environment in the molecule. Magnetic field-induced

circulation of electrons in a molecule induces a shielding field, which opposes the

extemal magnetic field. The induced field arkes from both electrons associated with the

atom and electmnr in neighbouring atoms and bonds. As a result of this shielding field, a

particular nucleus in a molecule will give a NMR signal at a lower fiequency than for an

isolated nucleus with no surrounding electrom. For historical reasons, a nucleus giving a

signal at relatively low fiequency is said to be shielded, while one giWig a signal at

relatively higher fiequency is said to be deshielded. This reflects the fact that early NMR

spectrometers operated at constant fiequency while varying the magnetic field. By

contrat, modem spectrometers operate at constant field and mesure fiequency

differences.

Because the Larmor fiequency is proportional to the strength of the magnetic

field, there is no absolute scale of chemical shiR Thus, a freguency difference (Hz) is

measured fiom the resonance of a reference compound, normally tetramethylsilane

(TMS) for 'H and I3c NMR, and divided by the absolute value of the Larmor fiequency

of the reference compound, which itself is proportional to the strength of the magnetic

field. TMS is generally useful because it is chernically inert, symmeaical, soluble in most

organic solvents, and absorbs at higher field (shielded) than almost al1 protons of organic

molecules. The chemical shifi is therefore given in the dimensionless scale of parts per

million (ppm, 6 scale), because a fiequency difference in Hz is divided by a fiequency in

MHz, these values are in a proportion of 1 : 1 06.

The local electronic environment normally dominates the chemical shift of a

nucleus. However, the anisotropic magnetic susceptibility of an adjacent bond may

influence the chemical shift of a nucleus as well. For example, the carbon-carbon bond is

the axis of a deshielding cone. Single bonds expenence shielding as in the case of

cyclohexane (See Figure 1-2). Here, a difference of about 0.5 ppm exists between the

resonance frequencies of the axial and equatorial protons. The equatorial 'H, which is in

the deshielding cone, is ofien more deshielded than the axial 'H on the sarne carbon. This

differential influence on the resonance is an important aid in confornational analysis of

compounds.

Figure 1-2 - Effect on equatorial (eq) and axial (ax) protons caused by a desbielding cone in six membered rings

This magnetic anisotropic effect can occur in any chemical group such as a

carbonyl (See Fijyre 1-3), where the circulation of electrons is less restricted about one

molecular axis than the others. The diamagnetic shielding of a proton by its 1s electron

density is relatively small compared with the shielding of nuclei of heavier atoms that

have filled inner shells. The proton near the functional groups experiences both high-

field and low-field shifts depending on theu position with respect to the anisotropic

group. Bemene protons have a chemical shift of -7.3 ppm, which is caused by

deshielding by a ring current of x electrons.

Figure 1-3 - The anisotropic shielding cones for a carbonyl group. Anisotropy causes deshielding of protons lying in thc cone whose a h is along the C=O bond and sheildiog outside this cone

1.4. Multiolicitv and Cou~ling 10,ll

Indirect or scalar coupling of nuclear spins through covalent bonds causes the

splitting of NMR signais into multiplets in high-resolution NMR spectroscopy in the

solution state. The signai multiplicity is the extent to which a NMR signal is split as a

result of spin-spin coupling. Signals that show no splitting are known as singlets (s).

Those with two, three, four, five, six or seven lines are known as doublets (d), triplets (t),

quartets (q, Figure 1-4), quintets (qui), sextets (sxt) and septets (sep) respectively. These

multiplet lines, however, must be equal distance apart to ensure that the one coupling

constant is shared by al1 of them. When m o or three different coupling constants

produce a multiplet, this is referred to as a two or three-fold multiplet, respectively [e.g. a

doublet of doublets (dd) or a doublet of doublets of doublets (ddd), Figure 1-41.

Qu artet ( q) Doublet of doublets (dd) Pseudotriplet Threefold doublet (ddd)

Figure 1-4 - Quartet, doublet of doublets, pseudotriplet and three-fold doublet (doublet of doublets of doublets)

The coupling constant is the frequency difference J i n Hz between two multiplet

lines. Unlike chernicd shift, the fiequency value of a coupling constant does not depend

on the strength of the rnagnetic field. In high-resolution NMR a distinction is made

between coupling through one bond ('JCH), and coupling through several bonds, e.g. two

bonds ( 2 ~ C ~ , gerninal couplings), ihree bonds (3~CH, vicinal couplings) or four or five

1 bonds (4~cH or 5.JCH, long-range couplings). The one-bond coupiing constant, JCHi is

proportional to the s character of the hybrid bonding orbital of the coupling carbon atom

(Le. '.JCH =500xs, where s = 0.5, 0.33 and 0.25 for sp, sp2, and sp3 hybridized carbon

atoms respectively). ' J ~ ~ increases with the electronegativity of the substituent bound to

the coupled carbon atoms. Two bond couplings, 2 ~ C H , across carbons of sp2 or sp3

hybridization are usually small with the exception of coupling across a carbonyl group.

Vicinal CH coupling constants, ' J ~ , resemble '.JHH in their dependence on the dihedral

angle between the C-C bond to the coupled C atom and the CH bond to the coupled

proton. An electronegative substituent on the coupled C raises the 3 ~ C H while one on the

coupling path lowers it.

1 .S. Instrumentation Ex~erimental

Mass spectral data (hi& resolution and low-resolution electron, EI) were

detemined at an ionizing voltage of 70 eV at 250°C on a Micromass 70-250 S (double

focusing) mass spectrometer. Optical rotations were measured on a Perkin-Elmer 341

polarimeter in CHC13 solutions. Fourier transfomed infrared (FTIR) spectra were

recorded on a Perkin Elmer Pentagon 500 instrument, using KBr discs. Melting points

were determined on Thomas-Kofler micro hot stage and are corrected.

Routine 'H and I3c NMR spectra were obtained on a Gemini-300 or Varian

üNITY-400 spectrometer equipped with a four-nucleus computer switchable probe while

2D experiments were carried out on a Varian UNITY-500 spectrometer equipped with a

5-mm inverse detection probe. The sarnples were dissolved in 0.8 ml of deuterated

solvent (-2-1 5 rnd0.8 ml solvent). COSY, NOESY, ROESY and HMBC experiments

were carried out using standard Varian software. HSQC spectra were obtained using

12 software developed in our laboratories. NOESY, ROESY and HSQC spectra were

acquired in phase sensitive mode, while HMBC was processed using mixed mode (phase

sensitive alongfi, absolute value alongfr). A mixing tirne of 0.1 - 1.25 s were used for

ROESY experiments. Linear prediction was used for al1 2D spectra except absolute

value COSY spectra.

Chaprer 2. Structural Elucidation of a Svnthetic Intermediate

2.1. Backeround

The first NMR investigation was used to elucidate the structure of an unknown

side product obtained in a previous attempted synthesis of aspidospermidine in our

laboratory. l 3 NMR methods and mass spectrometry were used to elucidate the structure.

Scheme 2-1 shows the synthetic steps taken to form the aspidospermidine precursor, S.

The key step, the photocyclization of azide 4 failed, but a small amount of an apparent

byproduct was obtained fiom one attempt. Elucidation of this product was of interest,

because understanding what intermediate was produced may explain what mechanism

had actually taken place in this photosynthetic reaction. Examination of this byproduct

eliminated the possibility that it was the desired product, 5. However, instrumental

limitations prevented a complete elucidation of this product. The structure of this

cornpound remained a mystery and this appeared to be an excellent subject for study

using our present NMR methods.

Figure ; C-1- Structure of aspidospermine

2 aq. acetone 1 NaN,

"J3

5 Desired Product

Scheme 2-1 - Synthetic steps peiiormed in attempt to synthesize precursor to Aspidospermidine, 5.

2.2. NMR data for Structure 3

Figure 2-2 - Structure 3

I3c, 'H NMR, 'H-'H COSY and HMBC 500 MHz NMR data obtained are shown

in Table 2- 1.

Table 2-1 - 500 MHz NMR data for 3

Pos. I3c a ' H(J HZ) qb 'H-'H COSY HMBC 2 155.054 (C) 4,6

120.219 (CH) 127.789 (CH) 125.023 (CH) 121 .O95 (CH) 141.925 (C) 57.629 (C) 27.292 (CH2)

7.67 (d, 7.7) 7.27 (dt, t .3,7.6) 7.12 (dt, 1 .O, 7.7) 7.04 (dd,0.5, 7.3)

1.18 (dd, 12.0, 12.5) 0.98 (ddJ1.8, 12.5) 2.74 (dd, 1.5,12.0) 1.96 (ddd, 6.1,l 1 .O,l2.8) 1.15 (m) 1 .O9 1.75 (dJ2.0) 1 .O8 (dd, 1 1 .O, 13 .O) 0.83 (dt, 4.7, 13.1) 1.8 1 (dd,3.0, 13.0)

4,5,6 3,596 3,4,6 3,435

lob, l lb, 12 10% llb, 12 16, l lb, 13 10,1la, 13 10, l lb , 13a 10,l la, 13a 12b, 13b 12b, 13a

5 6 3 4

3,5? 9 9, 10, 11, 13 10,l la, 12,13a

14 189.067 (C) 10,13a

a Chernicai shifts on 6 scaîe in 1 : 1 CDC13/c6D6 Multiplicity and coupling constants (in Hz) are in parenthesis. Pseudo axial position Pseudo equatorial position

2.4. Structural Determination of 3

Cornpound 3 was a white crystalline solid with a melting point of 243-245 OC. The

molecular formula, C20H28N2, was confimed by HRMS with a calculated molecular

weight of 368.2254 and experimental of 368.226849. This provided the fist piece of

evidence that the product was actually some form of a dimer. The 'H and I3c NMR

experiments in CDC13 gave evidence for 14 carbons and the correspondhg protons,

M e r providing evidence that the product was actually a symmetrical dimer. Since

there was overlapping of peaks of the 'H NMR in the aliphatic region, the expenment

was performed with increasing amounts of CsDs to resolve these peaks and determine

their coupling constants. Optimum resolution was found using a 1: 1 mixture of the two

deuterated solvents. COSY, HSQC and HMBC experiments were also performed using a

1: 1 mixture of CDCll and C6D6. These experiments were used to map together how this

molecule was attached.

Due to the molecular symmetry of the rnolecule, each side gave identical

chemical shifts and only half the nurnber of carbons and protons were evident. Carefùl

analysis indicated a cyclic ring attached to an indolenine. However, five distinct

methylene groups were present. The breakdown of the electron impact mass spectra with

molecular ion at 368 gave the next strong peak at 198, which would be the ion for 1 with

two CH2 attached. Additional breakdown peaks were at 184 and 170, which are due to

the loss of each of the methylenes. This suggested the existence of two six-membered

rings attached by two methylene groups at position 6, instead of two attached seven-

membered rings. Mapping of the 2-D NMR experiments determined the structure to be

3.

2.3 Iater~retation of Data

Since structure 3 is now established, it becomes clear that it is very unlikely that it

was produced in the photochemical reaction. Retracing the steps in the original synthesis

showed that 3 was actually an intemediate impurity fonned during the synthesis of 4. It

is almost certainly a byproduct fkom an earlier step.

In the first step of the synthetic sequence, carbazole 1 was treated first with ethyl

magnesium bromide in an anisole solution, and then with dibromoethane in a Grignard

reaction. Precautions were taken to minimize the possibility that both halogens of the 1,2

dibromoethane would react with 2 molecules of 1 to form 3. To form 4, the bromo

compound (2) was refluxed in an aqueous acetone solution mixture containing an excess

of sodium azide. Isolation and structural elucidation of this product, however, shows that

in fact some of the dibromoethane in the Grignard reaction did undergo a reaction where

each reacted with two molecules of 1. Despite purification, some of this product must

have been carried through to the photolytic step where it would not have been expected to

undergo m e r reactions. As a result, the structural question was resolved and the

possibility that an unknown photochemical reaction had taken place was conclusively

s h o w not to have taken place.

The stereochemical question, however, has not been resolved. The starthg

material was achiral, and there were no chiral constraints used to introduce the two stereo

centers at position 8 at each side of the dimer. Since only one set of chemical shifis was

observed, 3 should be either the racemic modification (RR, SS) or the meso modification

(W.

Chap fer 3 - Synthetic Plan of D ihydrocadambine

3.1. Previous svnthesis of 3a and 3B- dihvdrocadambine

3.1.1. Cadambine, 3a- and 3 0-~ihvdrocadambine'~ An extract of the heartwood of Anthocephalus cadamba first led to the isolation

of the indole glycosides, cadambine (7, and 3a-(8a) and Jfi-dihydrocadambine (8b).

These alkaloids are tryptamine derivatives with seven-membered rings. The structure

and relative configuration of 8a was assigned by McLean et al. as an alkaioid of Nauclea

diderrichds and later, by Brown and Fraser as an isolate fiom Anthocephalus ~adarnba'~.

Figure 3-1 - 7) cadambine; 8a) 3a-dihydrocadambine where 3H=a; 8b) 3p- dihydrocadambine where 3H=P

3.1.2. Previous synthesis"

OAc

OAc

Figure 3-2 - Intermediates used in the synthesis of 3a-dihydrocadrmbine.

A synthesis of 3a-dihydrocadarnbine (Ba) utilizing the glycol (10) denvative of the

tetraacetylated iridoid, secologanin (9), as a key intemediate, was previously performed

in our laboratory (See Figure 3-2). The first crucial step was the dihydroxylation reaction

of the vinyl group which was previously found to be highly stereoselective, but the yield

was low." This glycol was then quantitatively converted by the action of Amberlite IR

120 resin in methanol to 11. Treatment with dilute acid of 10 or 11 formed the

undesired-bridged bicyclic acetal 11. When instead, 11 was selectively oxidized with

pyridinium chlorochrornate at the primary alcohol it provided the aldehyde, 13. This key

intemediate locked the protective fiuiction of the cyclic acetal of both the secondary

hydroxyl group and the aldehyde. 13 was then reductively coupled with tryptarnine (14)

hydrochloride in the presence of sodium cyanoborohydride to form 15.

The completion of the synthesis of the alkaloidal framework then required

deprotection of the aldehyde funciion at C-5 of 15 and subsequent coupling with the

tryptamine moiety in an intramolecular Pictet-Spengler reaction. This proved difficult

due to acidic conditions needed and the susceptibility of the many cryptoaldehydes of the

iridoid structure. In addition, it was known that hydrolysis of the acetal was diflicult in

the presence of the neighbouring basic nitrogen. M e r extensive investigations, it was

found finally, that upon treatment with 90% formic acid, the target alkaloid was formed

as a mixture of 8a and 8b at high yield. The overall yield, however, was poor due to

instability of some key intermediates and the low yield obtained in the dihydroxylation.

This proved the Pictet-Spengler reaction could form a seven-membered ring in good yield

to form this alkaioid, but with M e stereoselectivity.

3.2. Svnthetic Plan

8 CHO

Scheme 3-1 - Biomimetic Retroynthetic Plan

Although the previous synthesis was successful in forming dihydrocadambine

(8a/8b), the sequence of reactions was only partiaily biomimetic because the strategy

essentially reversed the biosynthetic order of bond-forming reactions. A synthetic plan

was devised (See Scheme 3-1) to mimic the biogenetic order for which the desired

alkaloid is formed. A hypothesis for the biogenesis involves stereospecific oxidation of

the vinyl side chah of strictosidine (18) to an epoxide or its equivalent to form 16. l4 16

would then undergo an intemal opening of the terminal C-16 end of the epoxide by the

the sec amine nitrogen, closing the seven-membered Nig, and producing the secondary

alcohol. No matter what mechanism occurs to open the epoxide, an alkaloid would

form. Although the regioselectivity of the epoxide opening is uncertain, even if instead

the intemal opening of epoxide 16 occurred at the intemal C-15 end of the epoxide,

another alkaloid, is~dih~drocadambiw'~ (19a or 19b) would be formed.

Figure 3-3 - Structures of 18.) Strictosidine, where 3H=a 18b) Vincosidine, where 3H43 19a) 3a-Isodihydrocadambine, where 3H=a and 19b) 3 P- Isodihydrocadambine, where 3H=P

Strictosidine (180) itself, is a well-known monoterpene indole alkaloid glycoside

that is a precursor for over 2200 monoterpene indole and related alkaloids. 18a was first

isolated h m Rharya stricta by G.N. ~rnith.~' It was earlier thought that oniy vincoside

(18b) serves as a precursor for H-3a and H-3P indole alkaloid representatives of the

Corynanthe, Aspidosperma and Zboga fiameworks in Vinca ~ o s e a . ~ ' Strong proof of the

configuration of 18a had not been presented since it cannot be obtained in crystalline

f ~ r m . ' ~ It was found that only strictosidine and not vincoside is differentially

incorporated into several indole alkaioids such as those belonging to Corynanthe (3a and

3P series) Aspidospema and ~ b o ~ a ? Recent NMR studies have shown that the C-3

chiral center is in the S configuration, and thus it is strictosidine that serves as the major

precursor.24 In vivo, strictosidine is a biosynthetic equivalent of a Pictet-Spengler

condensation of a tryptarnine unit and secologanin (9), a reaction catalyzed by the

enzyme strictosidine synthase, which has been i~ola ted .~~

To form 16, a Pictet-Spengler condensation of the mono-epoxide of secologanin

(17) and the cornmercially available tryptamine hydrochloride could be perfomed. The

Pictet-Spengler reaction has long been an important procedure for the synthesis of both

indole and isoquinoline a lka l~ids~~, and in ideal conditions few side reactions are

reported. In the coupling reaction of tryptamine and secologanin, a new chiral center is

formed with complete stereoselectivity in the presence of the enzyme to form

strictosidine. In this synthetic sequence, it was hoped that the small amount of acid

present in the tryptamine hydrochloride would have three hctions. First, it could be

used to hydrolyze the dimethyl acetai to reveal the aldehyde group. Second, it could

catalyze the reaction of the aldehyde with the secondary amine of tryptamine to form an

imine, which is the start of the Pictet-Spengler reaction. Finally, it could also function to

protonate the imine to make it ~ ~ c i e n t l y electrophüic to do a substitution on the indole

(position 2) and complete the Pictet-Spengler.

A key step to this synthetic sequence is also the formation of the mono-epoxide,

17. The correct configuration of the epoxide is essential, first, to form 8a or 8b. In

addition, the configuration of the epoxide may have some infiuence on directing the

opening of the epoxide. Forming the correct isomer is known to be problernatic. Some

studies of the dihydroxylation and epoxidation of the vinyl side chah of the aldehyde-

protected form of secologanin (21) have previously been performed.27 Another iridoid,

sweroside (20), was used as a mode1 for the studies, but it was found that the two

derivatives behaved very differently in the oxidations carried out. 20 reacted site-

selectively with Os04 and m-chiorobenzoic acid at the vinyl side chah, leading to an

epimeric pair of products (di01 and epoxide, respectively), but with little

stereoselectively. 21 reacted at both the endocyclic double bond (a P-allcoxyacrylate) and

the vinyl side chah with little apparent site selectivity, low yield, but with very high

stereoselectivity to fom the desired configuration for this ~ynthesis.*~

Figure 3-4 - 20) tetraacetylated Sweroside and 21) aldebyde protected dimethyl acetal of 9 (secologaain)

It is hypothesized that either stenc factors or the conformation of the vinyl side-

chain are responsible for the reactivity of the Palkoxyacrylate of 21. The carboxyl

function of 20 is part of a lactone, and the carbonyl is locked in a conformation where its

n orbital interacts with that of the C=C, withdrawing electron density and deactivating it

to electrophilic attack. In the case of the 21, the ester can rotate so that the carbonyl x

orbital is not lined up with that of the C=C. With 21, the ring is more flexible since it is

not fused to the lactone ring. It readily places the side chain in an equatorial

environment, where there is considerably more stenc congestion, and the two faces of the

carbon double bond are quite different in the stenc himirance experienced by an

approaching reagent.

The low degree of stereoselectivity at the vinyl side chai. of 20 can be explained

by postulathg that this side chah is held in an axial coaformation in al1 derivatives of 20

(see Figure 3-5). In this conformation there is little difference beiween the two faces of

the C=C as far as stenc congestion is concemed. In 21, the ring is more flexible since it

is not fused to the lactone ring, and it readily places the side chah in an equatorial

environment. In an equatorial environment, there is considerably more steric congestion,

and the two faces of the C=C are quite differed in the steric hindrance experienced by an

approac hing reagent.

axial

Swerosi de 20

Figure 3-5 -Positioning of olefinic side-chah in sweroside(20) and secologanin(21)

Another anticipated problem in the synthetic plan, was choosing the optimal

acidic conditions to deprotect the diacetal protecting the aldehyde of epoxide 17.

Deprotection of acetals requires acidic c0nditions.2~ The glucose unit contains an acetal

rnoiety linking it to the secologanh aglycone, and the aglycone contains two "crypto-

aldehyde" units (acetai or related function). Al1 of these cm undergo acid-catalyzed

cleavage and thus the challenge lies in finding acid conditions that will cause the desired

reactions to take place, but leave the glucose etc. intact.

Chapter 4. Isolations fronr Lonicera tartarica

The synthesis of 3a- or 3P-dihydrocadambine requires the use of the natural

product, secologanin (9), as a synthetic precursor. It was known that secologanin cm be

isolated fiom the common honeysuckle plant, Lonicera tartarica. The first task was to

isolate this natural product from its known naturai source.

4.1. Iridoids of Lonicera tartarica

Secologanin, a monoterpenoid glycoside, is a precursor of more than 2200

alkaloids. It has been used as an insect repellant and is a nototious stimulator of

The compound is exceptionally rich in functional groups and chernical reactions can be

carried out at al1 carbon atoms of the aglycone skeleton.

Secologanin was first theoretically elucidated as a result of biosynthetic studies on

the rnonoterpenoid indole alkaloids3', which first established loganin (22) as the key

intermediate and it was then predicted that secologanin should be the last non-

nitrogenous key intexmediate to form the protoalkaloids.32 Secologanin is the ultimate

precursor for the C-9lC-10 non-tryptamhe carbon skeleton common to the majority of

indole alkaloids. 33

A related irîdoid, sweroside (20) can also be isolated fiom Lonicera tartarica.

Sweroside is a close relative of secologanin, but with the aidehyde function of

secologanin reduced to the level of an alcohol and incorporated into a lactone.

Secologanin and sweroside are cailed secoîridoids because they are biogeneticaily formed

from loganin, an iridoid glucoside, by cleavage of its cyclopentane ring?4

Column chromatography was performed using BDH silica gel (40-63 pm).

Charcoal and celite were obtained fiom Fisher Chernical Co. Pre-made preparative thin

layer chromatography (thickness of 0.25mm, lm) containhg a fluorescent indicator

(254 nm) were obtained fiom Toronto Research Chemicals. Reagent grade solvents were

used for al1 preparative chrornatography. Analytical thin layer chromatography (TLC)

analyses were c h e d out using Whatman polyester or aluminum backed plated precoated

with silica gel UV2s4 (0.25mm). TLC were viewed under ultraviolet light (254 and 366

nm) before spraying with phosphomolybdic acidkeric sulphate spray made up with

phosphomolybdic acid (4g), cenc sulphate (0.5 g) in 100 ml of a 20% &SO4 solution.

4.3. Isolation of Secologanin from Lonicera tartarica

Shoots of Lonicera tarturica (the common honeysuckle plant), bearing both pink

and yellow flowers were gathered from Su~ybrook hospitai by permission. The plants

were gathered the first week of June of 1997, while plant flowers were just begiming to

die.

The shoots (-5 kg) were broken manually Uito smaller pieces. The broken shoots

were left to soak in rnethanol approximately 2 hours. A Waring blender (2-litre capacity)

was then used at low speed to blend the mixture. A total of Idlitres of methanol was

used for soaiing and blending. The aqueoudmethanol extract was filtered through

Whatman filter paper and concentrated by mtaevaporation. The remaining filter cake

was saved for fûrther extraction, repeating the previous steps of soaking, blending and

filtering. Al1 extracts were collected together to form a thick greenhlack liquid.

The concentrate was extracted with 5 x petroleum ether or hexanes. The aqueous

solution was once again concentrated by rotaevaporation to collect approximately Mitre.

The aqueous solution was extracted successively with chloroform and ethyl acetate. The

ethyl acetate-soluble residue contained the most products by mas, with one compound

corresponding to the Rfof the desired product, secologanin, by TLC. This solution was

run through a celite/activated coconut charcoal column (300g/700g) which was set with

methanol. The solution obtained was an amber brown colour. The crude mixture was

acetylated by treatment with acetic anhydride in pyridine to protect the free hydroxyl

groups. Silica column chromatography was perfonned using various eluents to puri@ the .

products. Three main products 9,20 and 22 were obtained and structurally examined.

4.4. Products Isolated from Extract

Figure 4-1 - Structures 9,20 and 22

4.4.1. Identification of 22

5 mg of compound 22 was isolated as oil and examined by 'H, I3c, I H - I H COSY

NMR and HSQC expenments. NMR data is show in Table 4-1.

Table 4-1 - 400 MHz NMR data for 22

Pos. 13C a 1 ' H - I H COSY

1 97.7 (CH) 5.1 1 (d, 4.6) 2 7 - 43.8 (CH) 2.5 13 (m) 1 .3 ,7

3 42.7 (CH) 1.81 (ddd, 4.6,6.8,9.3 ) 2,4,5

4 13.4 (CH3) 1 .O7 (d, 6.8) 3

5 75.1 (CH) 4.02 (m) 3,6a, 6b

6a 42.2 (CH2) 2.23 (ddd, 1.6, 7.9, 14.2) 5,6b, 7

6b - 3.94 (dd, 1.3, 10.1) 5,6a, 7 7 46.5 (CH) 3.10 (m) 6a, 6b

8 114.1 (C) - - 9 152.2 (CH) 7.38 (d, 1.3) 1

' Chernical shif€s on 6 scale in CD30D In brackets, mdtiplicities and coupling constants (Hz)

11 51.5 (CH3) 3.68 (s)

1 ' 100.2 (CH) 4.72 (d, 7.6)

2 ' 74.6 (CH) 3.30 (dd, 7.6,9.7)

3 ' 78.2 (CH) 3.55 (t, 9.7) 2'' 4'

4' 7 1.3 (CH) 3.18 (t, 9.7) 3'' 5'

5 ' 77.8 (CH) 3.25 (m) 4'' d'a, 6'b

6'a 62.1 (CH2) 3.52 (dd, 9.6, 10.1) 5'' 6'b

6'b - 4.15 (dd, 4.5, 10.1) 5', 6'a

Compound 22 had a molecular ion peak at 600, and the high-resolution mass

spectrurn confirmed the molecular formula as C2,H350 Compound 22 was identified

as loganin pentaacetate fiom examination of NMR data and the structure and

configurations were confvmed by cornparison of data with reported values.35 Although

loganin is the known precursor to secologanin, which is abundant in Lonicera tartarica,

to the best of our knowledge, this is the fist time loganin has been isolated fiom this

plant.

a Note: The NMR experiments performed could not determine exact positions of acetates

4.4.2 - Identification ef structure a 530 mg of compound 20 was isolated as a gurn fkom the extract. 'H, 13c and 'H-

'H COSY experiments were perfonned. The NMR data are as follows. 'H NMR (400

MHz, CDCb) 6: 1.68(rn, H-6a and H-6b), 1.97(s, Ac), 2.02(s, Ac), 2.04(s, Ac), 2.1 1 (s,

Ac), 2.70 (ddd, 1.5, 5.5, 9.7), 2.86(m, H-7), 3.75(ddd, 2.1, 4.0, 9.5, H-57, 4.14 (dd, 2.2,

12.4, H-6'a), 4.3 1 (dd, 4.8, 12.4, H-6'b), 4.32(m, H-Sb), 4.46(dt, 2.8, 10.8, H-5a), 4.92(d,

8.1, H-1'), 5.01(dd, 8.1, 9.7, H-2'), 5.10(t, 9.7, H-47, 5.25(t, 9.7, H-37, 5.28(m, H-4a),

5.3 1(m, H-4b), 5.46(ddd, 9.5, 10, 17.6, H-3), 7.55(d, 2.4, H-9). 13c NMR (125 MHz,

CDC13) 6: 20.6(CH3 for acetates), 24.7(C-6), 27.7(C-7), 42.2(C-2), 61.7(C-6'), 68.1(C-

4'), 68.5(C-5), 70.3(C-2'), 72.O(C-3 '), 72.5(C-S'), 96.O(C- 1 '), W.O(C- 1 ), 105 S(C-8),

i2OS(C-4), 13 1 S(C-3), 15 1.6(C-9), 165.7(C-1 O), 169.1, 169.5, 1 70.0, 1 70.6(C=O for

acetates). Compound 20 was confirmed to be the tetraacetate of sweroside. The positions

and configuration of protons and carbons were determined by comparing to previously

reported values.36 Compound 20 had a molecular ion peak at 526, and the high-

resohtion mass spectnun confimied the molecdar formula as C2,$H3()OI3.

4.4.3 - Determination structure 2 2.2 g of compound 9 was isolated as oil fiom the extract. 'H, "c, HSQC and

W C experiments were performed. The NMR data obtained are as follows. 'H NMR

(400 MHz, CDC13) 8: 9.70(t, 1.5, H-S), 7.42(d, 1.8, H-9), 5.46(ddd, 9.3, 10.1, 1 7.9,

5.28(dd, 2.1, 10.7, H-4a), 5.30(ddd, 2.1, 5.6, 17.1, H-4b), 5.23(t, 9.5, H-37, 5.10(t, 9.6),

5.02(dd, 8.1, 9.5, H-2'),4.89(d, 8.0, H-l'), 4.29(dd, 4.2, 12.4, H-6'a), 4.17(dd7 2.2, 12.4,

H-6'b), 3.74(m, H-S), 3.69(~, H-Il), 3.32(~, 0CH3), 3.30(s, OCHa), 2.94(ddd7 1.5, 6.2,

17.0, H-7), 2.8 1 (m, H-2), 2.42(dddY 1.2, 7.6, 18.0, H-6a), 1.54 (rn, H-6b), 2.1 1 (s, 6'-

Ac), 2.04(sy4'-AC), 2.01(s,3'-Ac), 1.91(s, 2'-Ac). 13c Nh4R (125 MHz, CDCli) 8:

20.6(CH3 for AC), 20.6(CH3 for 3', 4' and AC), 26.7(C-7), 42.2(C-2), 43.6(Cœ6)M,

5 1.3(C-Il), 5 1 .4(C-0CH3), 53.7(C-0CH3), 68.2(C-2'), 70.5(C-4'), 72.2(C-5')' 72.8(C-

3'), 95.7(C- 1 ), 95.9(C- 1 '), 96.1 (C-6'), 102.4(C-5), 1 1 1.4(C-8)' l2O.2(C-4), 132.2(C-3),

15 1.3(C-9), I69.O(C=O for AC), 169.5(C=O for A AC), 170.3(C=O for AC),

170.8(C=O for 6'Ac). The positions of carbons and protons were assigned fiom

HSQC and HMBC data. Compound 9 was confïmed to be the tetraacetate of

secologanin.

4.5. Forming dimethvl acetal of 9

The aldehyde function at position 5 of secologanin (9) was protected to prevent

oxidation and as a protective group against attack by various reagents such as strong or

moderately strong nucleophiles in subsequent synthetic reactions. This was done by

adding 9 (1.8g) to a solution of methanol and Arnberlite IR 120 resin as the source of acid

catalyst. The reaction mixture was then filtcred through celite and concentrated by

rotaevaporation to obtain 23 at quantitative yield.

More detailed structural elucidation was performed on the tetmacetylated

dimethyl acetal of secologanin (STAMA, 21) since this would be the precursor for most

of the synthetic reactions, and thus more knowledge was needed of the coupling constants

for each proton.

\

b I o 8

4 OAc

Figure 4-2 -Structure of 21 a) Stereo projection of STAMA; b) Chair representation of STAMA

The proton, carbon and 'H-'H COSY NMR assignments for structure 21 are

shown in Table 4-2. 'H, 13c, 'H-'H COSY, HSQC and NOESY experiments were

Table 4-20 500 M H z NMR data for 21

POS. I3c a 'H ab Coupling (Hz) 1 ~ - 1 ~ COSY 1 96.149 (CH) 5.30 (d) J12 = 4.4 2 2 43.222 (CH) 2.71 1 (m) 1,3,7 3 133.194 (CH) 5.608 (ddd) = 17.1 2,4a, 4b

JpCn = 10.7 J33 = 9.2

a Chernical shifis on 6 scde in CDC13 In brackets, multiplicities

Pos. I3c a 'H a,b Coupling (Hz) 'H-'H COSY 120.153 (CH2) 5.260 (ddd) 3,4b 4a

4b

5

6a

6b 7 8 9 1 ' 2'

3 '

4'

5'

6'a

6'b

2' Ac Acetate 3' Ac Acetate 4'-AC Acetate 6'Ac Acetate Acetal Acetal

102.442 (CH)

- 27.304 (CH) 1 1 1.406 (C) 150.327 (CH) 95.755 (CH) 70.576 (CH)

72.499 (CH)

68.143 (CH)

72.166 (CH)

5.297 (ddd)

4.479 (dd)

1.540 (ddd)

2.170 (m) 2.787 (m)

7.320 (d) 4.873 (d) 5.019 (dd)

5.210 (dd)

5.1 O7 (dd)

3.726 (ddd)

4.138 (dd)

4.287 (dd)

2.029 (s)

2.003 (s)

1.926 (s)

2.104 (s)

3.309 (s) 3.299 (s)

3,4a

da, 6b

5,6b, 7

5'6% 7 2,7,6a, 60

7 2' l' , 3'

2'' 4'

3'' 5'

4', 6'a, 6'b

5', 6'b

5'' 6'a

(C=O)OCH3 66.983 (CH3) 3.700 (s)

The molecular formula of 23, C27&00L7, was confinned by HRMS. IR confïrmed

the presence of ester carbonyl at 1756 cm-' also the vinyl ether at 1228 cm'. The 'H-'H

COSY spectrum confmed the relative positioning of al1 protons. H d a and H-6b showed

different chernical shifis, but the COSY cross-section shows H-6a and H-6b showed cross

peaks with each other, H-5 and H-7, while H-5 and H-7 showed no cross peaks with each

other. Examination of HSQC spectra provided the correlation for directly bonded proton

and carbon pairs.

The reactions performed in an attempt to accomplish the synthesis of

dihydrocadambine (8a or Sb) are discussed in this chapter.

5.1. mCPBA oxidation of 8

A peracid oxidation of STAMA (21) was performed to see if conditions could be

optimized to form the mono-epoxide at the vinyl position.

Figure 5-1 - Structure of bis-epoxide 23

5.1.2. General ~rocedure for ~eracid oxidation of STAMA (21)

To a solution of olefin 21 (C27H380is; 150 mg, 0.25mmol) in dichloromethane

(hl) at 0°C was added mCPBA (150mg, 3 equiv.). AAer an hou, the ice bath was

removed and the mixture was shed at room temperature for 72 hours. 5% NaHC03

(20rnl) was added and the mixture was extracted with dichloromethane ( 2 x ) . The organic

phase was then washed with brine, dried over MgS04 and concentrated by

rotaevaporation. 2 products were visible by TLC. Chromatography on silica using 9: 1

CHC13/acetone was performed. The main product was obtained at 50% yield as the bis-

epoxide 23, with a mp of 153-1 55.5 OC. 'H NMR (300 MHz, CDC13) 65.082 (H-1, d,

1 .a), 1 -623 (H-2, ddd, 10.0, 7.8, 1.8), 3.272 (H-3, 6.76, m), 2.47 (H-4a, 2.777 (H-4b, dd,

4.5,3.6) 4.672 (H-5, t, 5.5) 1.89 1 (H-6% m) 1.801 (H-6b, m) 3.172 (H-7, m), 4.853 (H-

9, s), 3.776 (CH30-IO , s, 3), 3.301 (CH30-acetal, s, 3), 3.340 (CH30-acetal, s, 3),

4.921 (H-l', d, 8.1), 5.013 (H-2', t, 9.3), 5.203 (H-3', dd, 9.4, 9.7), 5.105 (H-4', t,

9.7), 3.862 (H-5', m), 4.167 (H-6'a, dd, l2.5,2.3), 4.820 (H-6'b, dd, l2.5,4.6), 2.1034

(Ac, s, 3H), 2.033 (Ac, s, 3H),2.010(Ac, s, 3H), 1.954 (Ac, s, 3H).

Another product was obtained at 30% yield, which appeared to be oxidized at the

P-alkoxy acrylate bond and not the vinyl bond, because of the disappearance of the H-9

proton by NMR while the H-3 and H-4 vinyl protons were retained. Prolonged mixing

and heating of this product only resulted in the production of more of the bis-epoxide

(23) indicating that the 8-alkoxy acrylate bond was actually the first to be oxidized.

5.2. Dihvdroxvlation of STAMA 37,38

The oxidation of olefms is known to selectively perform dihydroxylation

reactions at high yields using osmium tetraoxide (0~0~). Although 0 ~ 0 4 , like the

peracids, is selective for less polarized double bonds with more electron donating

substituents, the selectivity has been shown to be greate?9 and the influence of steric

factors seems to be stronger than in the peracid oxidations.

To prepare the 1% 0s04 solution in pyridine, reagent grade Os04 (99+% pure)

was obtained in 1 g glass ampoules which could be scored with a file, cooled in Dry Ice

and broken open. The orange solid was quickly transferred to an amber vial that had

been pre-cooled, with septum under argon gas. The &O4 was dissolved in 10 ml of

pyridine .

A catalytic amount of osmium tetraoxide ushg N-methylmorpholine N-oxide

hydrate (NM0.H20) as a CO-catalyst was used. Reactions were carried using Os04

dissolved in pyridine or tert-butyl alcohol.

To a solution of STAMA (21) (C27H3(015; I.Og, 1.7 rnrnol) and N-

methylmorpholine N-oxide hydrate (NMO-H20, 0.48g, 2.1 equivalents) in acetone (12

ml) and water (1.2 ml) at room temperature was added a solution of Os04 (1 -2 ml of a 1 %

solution in pyridine. The mixture was stirred at room temperature for 48 hours. A

saturated solution of Na2S03 (20 ml) was then added to the resulting mixture, and

continued stimng for 2 hours. The mixture was extracted with ethyl acetate ( 2 x ) and the

organic phase was then washed successively with saturated Na2S03. water, and brine and

then dned over MgS04 and concentrated d o m by rotaevaporation. Flash

chromatography using 4: 1 CHC13/acetone was performed to isolate the product. A solid

was obtained at 79% yield.

5.2.1. NMR Examination a glvcol24 The proton and deduced carbon NMR assignments for glycol 24 are shown in

Table 5-1. 'H, HSQC, COSY and ROESY experiments were performed in CDCl,.

Table 5-1 - 400 MHz "C and 'H NMR Data for glycol 24

Pos. J values 9 7.326 (d) 1.5 151.2

3.001 (m)

1.885 (m)

1 S60 (m)

4.576 (dd)

3.560 (bs)

3.860 (bs)

2.810 (m)

5.302 (d)

4.923 (d)

5.024 (t)

5.259 (d)

5.107 (t)

3.716 (m)

4.265 (dd)

4.199 (dd)

Acetd 3.410 (s,3)

Acetal 3.264 (s,3)

Acetates 2.1 OS(s,3), 2.049(~,3), 2.025(~,3) and 2.046 (s,3)

Glycol 24 was obtained as a white crystalline solid with a melting point of 84-

86°C. The carbon NMR assignments shown in Table 5-1 were al1 deduced fiom an

a Chernical shifts on 6 scale in CDC13 Al1 carbon shifts were determined by HSQC

HSQC experiment. COSY data was used to confimi positions of ail protons.

5.3. Formation of kev epoxide (27)

Scheme 5-10 Conversion of glycol 24 to epoùde 26

5.3.1. Conversion glwoi 24 to e~oxide 26

nie glycol (24) could now be used to form the monospoxide of 21, using the

steps outlined in Scheme 5-1. This sequence of reactions was perforrned in two steps,

without intermediate cation of the tosylate (25).

Glycol 24 (C27&0017; 400mg, 0.6 rnrnol) was dissolved in dry pyridine (20 ml)

with freshly regenerated 4A molecular sieves, and cooled to 0°C. pToluenesulfony1

chloride (1 30 mg, 1 .1 equiv.) was added as a solid and the resulting mixture was stirred at

0°C for an additional 2 hours and then gradually warmed to room temperature. After

stimng at room temperature for 24 ~ O W S , 5% HCI was added and the resulting mixture

was extractcd with EtOAc (2x). The organic layer was washed with brine, dried over

MgS04 and concentrated. This residual oil was mixed with K2C03 (100 mg, 2.1 equiv.)

in methanol (70 ml) and was stirred at room temperature for 48 minutes, and then

saturated N h C l was added. The mixture was extracted with EtOAc (2x) and the organic

layers were washed with brine, dried over MgS04 and concentrated. The oil was purified

by silica chromatography using 9:l CHC13/acetone to obtain a solid at an overall yield of

70%.

5.3.2. NMR Examination of eooxide 26

Proton, 'H-IH-COSY, HSQC and ROESY experiments were performed and NMR

data are shown in Table 5-2.

Table 5-2 -500 MHz NMR of mono-epoxide 26

Labeled Proton #

J values

9 7.413 (d) 1.8 150.1 7 3.053 (ddd) 17.9,6.1, 5.4 42.4 6a 1.99 (m) - 43.10 6b 2.05 (m) - - 5 4.572 (dd) 3.6.7.6 105.1

a Chernical shifts on 6 scale in CDC13 MUltiplicities in parentheses Obtained fiom HSQC

Labeled IHB.~ J values 13cqc Proton # 4a 2.568 (dd) 4.8,2.6 47.9

5' 6'a 6'b (C=O)Oc& Acetal

Acetates

2.844 (m) 2.746 (m) 1.752 (m) 5.358 (d) 4.878 (d) 4.991 (t) 5.207 (t) 5.098 (t) 3.735 (ddd) 4.261 (dd) 4.162 (dd) 3.709 (s,3) 3.327 (s,3) 3.281 (s,3) 2.109,2.034,2.013 and 1.934

NMR analysis showed the main product to have the desired, R configuration.

5.4. Pictet-Spengler reaction

5.4.1. Cou~ling a STAMA J21) and trwtamine

Before attempting to perform the Pictet-Spengler condensation on mono-epoxide

26 and tryptamine hydrochloride, conditions needed to be optimized to see if the acid

present in the tryptamine could hydrolyze the protected aldehyde and immediately

cataiyze the Pictet-Spengler reaction. As a model, the reaction was performed on

STAMA (21) instead of epoxide 26 so that conditions could first be optimized. The

reaction was fist carried out using the method described by Battersby that uses

tryptamine hydrochloride.40 We were disappointed to find there was no hydrolysis of the

aldehyde and no reaction had taken place. Many conditions were then explored, using a

variety of acids, solvents and temperatures, but al1 of these were unsuccessN in

deprotection. No reaction was also obtained in one attempt to view if the use of epoxide

26 would yield diflerent results.

While exploring the various acidic environments, some side-products products

were obtained, and analyzed by NMR. The loss of the glucose residue was evident in

some cases, which was easily established fiom the losses of the acetates and sugar

protons by 'H NMR, and the concomitant isolation of pure acetylated glucose residue.

5.4.2. Coupling reaction of STA (9) and trv~tamine

In light of the problems deprotecting the dimethyl acetal of the aldehyde in situ, it

became obvious that epoxide 26 would fust have to be deprotected and then coupled with

tryptamine in two steps. Optimization of conditions to couple tryptamine was first

explored using the free aldehyde, secologanin (9) as a model. It was found that an

additional amount of a mild acid to that present in the tryptamine hydrochloride was

required for the reaction to proceed. Many conditions were explored, including catalytic

amounts of mineral acids, pTSA and acid resin.

5.4.2.1. General orocedure for cou~ling of 9 and trv~tamine

To a solution of 9 (C25H32014; 100 mg, 0.18mmol) in benzene (20 ml), water (1

ml) and acetic acid (0.5 mi) was added tryptamine hydrochloride (2.1 equiv., 75 mg).

The solution was heated at 50°C for 24 hours. The solution was then quenched in

ammonium acetate and extracted with EtOAc. The organic phase was then washed with

brine, dned over MgS04 and concentrated down by rotaevaporation. Flash

chromatography using 9:l CHC13/acetone was performed to isolate the product. Two

products, 18a and 27 were obtained.

5.4.3. Structure Determination of 18a

Less than 2 mg was obtained of compound 18a was isolated. NMR elucidation of

this product was performed using a microprobe to analyze the small amount of sample.

Figure 5-2 - Structure of 18a

'H, 13c, 'H -'H COSY, HSQC, HMBC and ROESY experiments were performed

on this molecule. Coupled HSQC was used to determine the coupling constants of many

of the overlapping protons. 'H, "C and HMBC data are shown in Table 5-3.

Table 5-3 -500 MHz "c, 'H and HMBC data for I8a

Pos. I3c a 1 ~ 8 . b

HMBC

0 133.1(C) 52.0 (CH) (NI 42.8 (CH2) - 20.9 (CH2) - 107.5 (C) 126.9 (C) 1 18.5 (CH) 120.1 (CH) 122.5 (CH) 11 1.8 (CH) 137.8 (C) 35.5 (CH2) - 32.4 (CH) 109.7 (C) 156.3 (CH) 1 19.4 (CH2) - 135.6 (CH) 45.2 (CH) 97.7 (CH) 171.1 (C=O) 52.3 (CH3) 93.9 (CH) 73.4 (CH) 72.5 (CH) 68.9 (CH) 71.7 (CH) 62.8 (CH2)

20.61 7 (CH3) 20.61 8 (CH3) 20.6 19 (CH3) 20.787 (CH3)

7.80 - 4.30 (ddd, 1.3,3.0, 11.4) 3.01 (s) 3.17 (ddd, 5.3,8.8, 12.3) 3.48 (ddd, 4.2,5.3, 12.3) 2.84 (ddd, 4.2,5.3,15.7) 2.96 (ddd, 5.3,8.7, 15.7) O

O

7.44 (d, 7.8) 6.99 (dt, 1.3, 7.4) 7.05 (dt, 1.1, 7.9) 7.30 (d, 7.7) - 2.09 (dd, 3.9, 14.7) 2.21 (dd, 1 1.5, 14.7) 3.05 (m) - 7.73 (d, 2.4) 5.24 (ddd, 1.0,2.1, 10.6) 5.35 (ddd, 1.0,2.1, 17.4) 5.79 (ddd, 7.6, 10.6, 17.4) 2.70 (ddd, 1.7,5.8,9.7) 5.73 (d, 4.4) - 3.78 4.90 (d, 8.1) 5.03 (dd, 8.1,9.7) 5.25 (t, 9.6) 5.11 (dd, 9.6,9.7) 3.77 (ddd, 2.1,6.1,9.7) 4.33 (dd, 6.1,ll.g) 4.1 1 (dd, 2.l,ll.g) 2.05 (s, 3) 2.07 (s, 3) 1.97 (s, 3) 2.13 (s, 3) -

Sa, 5b -

a Chernical shifls on 6 scale in CD30D In brackets, multiplicities and coupling constants in Hz

Pos. 13c a ' H " ~ HMBC

3' Ac 168.8(C=O) - 3 ' 4' Ac 167.6 (GO) - 4' 6' Ac 166.5 (GO) - 6 '

Not enough product was obtained to determine melting point or to obtain an IR

spectra. The results of the HSQC and HMBC experiments were used to detennine the

relative positioning of al1 carbons, nitrogens and protons. The results of the ROESY

experiment showed n.0.e. between H-15 and H-14a. H-3, however, only showed n.0.e.

with H- 14b and none with H-15. The stereochemical assignment for H-3 as the S isomer

was assigned on this basis. Bases on these results, the product was determined to be the

alkaloid, strictosidine.

5.4.4. Structure determination of 27

The main product, compound 27, was obtained at 79% yield fiom this reaction

9

H H' 14 16

H"

H

OAc

Figure 5 3 - Structure of 27

'H, 'k, 'H -'H COSY, HSQC, HMBC and ROESY experiments were performed

on this compound 27. Coupled HSQC was used to determine the coupling constants of

many of the overlapping protons. 'H, 13c, 'H -'H COSY and HMBC data are shown in

Table 5-2.

Table 5-4 500 MHz I3c, 'H, COSY and HMBC data for 27

Pos. 13c a ' H ' ~ ' H - ~ H COSY HMBC

(NI 132.693 (C) 52.853 (CH) 39.562 (CH2) - 20.948 (CH2) - 109.9 (C) 126.752 (C) 1 1 8.444 (CH) 1 19.905 (CH) 122.292 (CH) 1 10.884 (CH) 136.230 (C) 3 1.607 - 26.288 (CH) 108.306 (C) 146.835 (CH) 120.687 (CH2) - 13 1.754 (CH) 42.690 (CH) 96.064 (CH) 170.056 (C=O) 95.872 (CH) 70.461 (CH) 72.261 (CH) 68.133 (CH) 72.229 (CH) 61.723 (CH*) - 20.6 1 3 20.6 13 20.6 13 20.787

7.788 (br s) - 4.871 (d, 10.7) 5.165 (d, 11.9) 2.866 (dt, 4.5, 1 1.9) 2.805 (m) 2.805 (m) - - 7.509 (d, 7.7) 7.126 (td, 1.1, 7.9) 7.192 (td, 1.3, 7.4) 7.335 (d, 7.8) - 1.58 (ddd, 12, 13, 13) 2.18 (dddO, 4'4, 13) 2.94 (m) - 7.48 (d, 2.4) 5.18 (dd,1.7,10.1) 5.26 (dd, 1.7, 16.9) 5.47 (ddd (**) 2.70 (ddd, 1.8,5.8,9.7) 5.29 (d,4.4) - 4.91 (d, 8.9) 5.03 (dd, 8.9'9.7) 5.25 (t, 9.5) 5.11 (dd, 9.5,9.7) 3.77 (ddd, 2.4,4.4,9.7) 4.33 (dd, 4.4J2.8) 4.15 (dd, 2.4,12.8) 2.022 (s, 3) 2.044 (s, 3) 1.997 (s, 3) 2.111 (s, 3) -

- - 14% 14b 5b, 6 5% 6 5% 5b Sa, 5b - - IO, I l , 12 9, 11, 12 9, 10, 12 9, 10, 11 - 3, 14b, 15 3,14a, 15 14% 14b, 21 - - 18b, 19 18a, 19 18a,18b, 20 19,21 20 - 2' 1', 3' 2', 4' 3', 5' 4', 6'a, 6'b 5', 6'b 5', 6'a - - - -

' Chemicai shifts on 6 scaie in CDCll in brackets, multiplicities and coupling constants in HZ Pseudo axial position Pseudo equatorial position

Pos. 13c a ' H " ~ WH COSY HMBC

5.4.4.1. Structural Determination of 27

The product was obtained as an orange-brown solid with an [a], = -45 O . High-

resolution mass spectral anaiysis confirmed the molecular formula of C34H38N2012,

which has a calculated mass of 666.24254 and an experimental mass of 666.243061. The

infrared spectmn confirmed absorption due to primary amine (3378 cm-'), carbonyl

(1 755 cm-'), vinyl ether (1 228 cm-') and disubstituted aromatic (755 cm").

The 'H NMR resonance peak at 6 7.788 (N-1) showed evidence that the indole

NH remained intact. There was no amine peak indicated for position 4 in the 1 H-NMR,

indicating that this nitrogen had no attached protons as strictosidine (18a) did. In

addition, there were no HMBC correlation's of 639.562 (C-5) with any protons except

those at C-6. No geminal coupling exists for 6a and 6b since they essentially have the

same chemical shift. The "C resonance's for the lactam carbonyl at 5170.056 and the

sugar carbonyls 6 170.03 1 (2') 169.742 (3') 169.506 (4') 170.622 (6') and the lactam

were similar, however HMBC clearly distinguished the positioning of dl.

Interestingly, the two-gerninal protons at position 5 had two very different

chemical shifts of 2.886 and 5.165 ppm. The ciifferences in sh ih present here are due to

the anisotropic effects caused by the amide. The positioning of these protons was evident

by the HSQC contours and the ')c cross-section of the coupled HSQC spectrurn, which

showed thwe two protons, attached to C-5. Further evidence that both these protons were

attached to C-5 came fiom the COSY spectnun, which showed cross-peaks between H-Sa

and H-Sb were present with Hda and H-6b.

The configuration at C-3 was deteminrd by a NOESY experiment, which

showed that the proton was on the same face as H-14b and H-15. The product obtained

had 3R-configuration. The NOESY enhancements are shown in Figure 5-4.

Figure 5-1 - nOe enhancements observed for Compound 27

The designation of the 3R-configuration is the reason it was assigned as

vincosamide and not stricto~amide.~~ Vincosamide has been show as a precursor in the

biogenetic route to the synthesis of the N d e a akaloid, nauclefidine" and naucleudinal.

(Figure S-S) .~~~" Nauclefidine has been found to exhibit potent andgesic and antibacterial

activities.

Nauclefidine Naucleidinai

Figure 5-5 - Structure of Nauclefidine and naucleudinal

5.5. Deprotection of dimethvl acetal of 26

Since the coupling to tryptamine had to take place in two steps, epoxide 26 had to

be converted to its deprotected ddehyde. Before deprotecting the diacetal of epoxide 26,

STAMA (21) was used as a mode1 to explore optimum conditions. The problem proved

to be more problematic than anticipated. It was first hoped that mixing of 23 in a 1: 1

benzenelwater mixture using acid resin as a cataiyst would be suficient for the

deprotection. The results were unsatisfactory and other routes to deprotection were

investigated. In one attempt to optimize the coupling of tryptamine to 23, trimethylsilyl

trifluoro methane sulfonate (TMSOTf) was used as a ~ a t a l ~ s t . ~ ~ It was found instead that

these reaction conditions successfully deprotected the dirnethyl acetal of 23. These

conditions were then applied to epoxide 26.

To a solution of epoxide 26 (CZ7&&; 100 mg, 0.18mmol) in 10 ml dry

tetrahydrofuran -20°C under argon gas, was added TMSOTf (2 equiv., 120 kL). The

reaction was stirred for 45 min and 1 ml of water was added. The reaction mixture was

then quenched in ammonium acetate and extracted (3x) with ethyl acetate. The reaction

mixture was dned over MgSOd and concentrated. Silica chromatography using 9:l

CHC13/acetone was perforrned to isolate epoxide 28 at 80% yield.

Figure 5-6 - Structure and proton 400 MIIz NMR assignments for epoxide 28.

The proton assignments for 28 are show in Figure 5-6. Proton and 'H-'H COSY

NMR experiments confirmed that the desired fiee aidehyde epoxide derivative of

secologanin (28) needed to couple to tryptamine in the biomimetic synthesis of

dihydrocadambine (8d8b) was successfully formed.

5.6. Cou~ling of ewxide 28 to tw~tamine

Epoxide 28 was then coupled to tryptamine hydrochloride in a benzene/water

solution. Compound 29 was obtained at 60% yield.

Figure 5-7 - Structure of compound 29

'H, 13c, 'H -'H COSY, HSQC, W C and ROESY experiments were performed

on this compound 28. Coupled HSQC was used to determine the coupling constants of

many of the overlapping protons. 'H, "c, 'H - l ~ COSY and HMBC data are shown in

Table 5-5.

Table 5-5 - 500 MHz NMR 'k, 'H and HMBC data for 29

Pos. 13c a ' H " ~ HMBC

(NI 131.8(C) 53.4(CH) 39.6 (CH2) - 20.9 (CH2) - 1 10.1 (C) 126.5 (C) 131.1 (CH) 122.1 (CH) 1 19.7 (CH) 1 18.5 (CH) 137.2 (C) 31.91 (CH2) - 26.92 (CH) 109.7 (C) 156.3 (CH) 40.9 (CH2) - 48.82 (CH) 45.2 (CH) 90.25 (CH) 171.1 (C=O) 95.76 (CH) 72.3 (CH) 70.7 (CH) 69.1 (CH) 67.9 (CH) 62.8 (CH2) - 20.61 (CH3) 20.62 (CH3) 20.62 (CH3) 20.8 (CH3)

8.05 (s) - 4.756 (d, 11.4) 5.13 (d, 12.5) 2.86 (m) 2.79 (d4,6.8,12.7) 2.10 (m) - - 7.51 (d, 7.8) 7.138 (dt, 1.3, 7.4) 7.1 1 (dt, 1.3,7.9) 7.14 (d, 7.7) - 2.44 (d, 13.6) 1.80 (m) 3.10 (m) - 7.73 (d, 2.4) 2.34 (d, 6.8) 2.58 (m) 2.88 (m) 2.70 (ddd, 1.7, 5.8,9.7) 5.47 (d, 4.4) - 4.87 (d, 8.3) 5.26 (dd, 8.3,9.7) 5.25 (dd, 9.6'9.7) 5.10 (dd, 9.6'9.7) 3.78 (ddd, 2.1,6.1,9.7) 4.33 (dd, 6.1,11.9) 4.11 (dd, 2.1,11.9) 2.04 (s, 3) 2.07 (s, 3) 1.97 (s, 3) 2.12 (s, 3) -

-

' Chernicd shifts on b scale in CDC13 In brackets, multiplicities and coupling constants in Hz

Pos. 'Y a ' H ' ~ HMBC

The resuits of HSQC and HMBC experirnents for compound 29 were used to determine

relative position of atoms. The configuration around H-3 was determined to be R upon

examination of ROESY expenments. The results show that lactamization occmed under

these conditions also to f o n this epoxide denvative of vincosamide.

5.6. Discussion of results

The lactarns 27 and 29 were the major products formed in the Pictet-Spengler

reaction attempts. This demonstrates that in acidic conditions, the nucleophilic nitrogen

preferentially attacks the sp2 carbon of the ester over the sp3 of the epoxide. Once it was

realized that acid media caused attack of the methyl ester in the coupling of tt-yptarnine to

secologanin derivatives, attempts were made to perform a Pictet-Spengler reaction in

aprotic media using methods described by Cook et without success. The

formation of an epoxide derivative of strictosidine (16) was more problematic than

anticipated. Conditions need to be found to successfully complete the Pictet-Spengler

reaction of epoxide 27 to tryptamine in conditions that will prevent the lactarnization of

the ester. The reaction may also be successful if the ester in epoxide 27 could be

converted to its acid denvative to make this carbonyl Iess electrophilic and successfully

couple this derivative to tryptamine.

Chapter 6. NMR Analysis of Natuai Products

NMR methods were also used to elucidate the structure of unknown naturd

products. Al1 of the natural products analyzed in this chapter were done in collaboration

with scientists from the University of West Indies campuses in Barbados and Jamaica.

The samples exarnined in this chapter were isolated and purified in the West Indies, and

the NMR data were performed at the University of Toronto.

6.1. Terpenes 48

Terpenes (also referred to as isoprenoids) are naturally occurring compounds

having isoprene (C5Hs) skeletal units (see figure 2-1). Terpenes rnay contain two, three,

or more isoprene units. Their molecules may be open chah or cyclic. The number of

isoprene units they contain classifies terpenes as follows:

Monoterpene 2 isoprene units 1 10 carbons

Sesquiterpene 3 isoprene units / 15 carbons

Diterpene 4 isoprene units / 20 carbons

Triterpene 6 isoprene units / 30 carbons

Tetraterpene 8 isoprene units 1 40 carbons

Most of the compounds discussed in this chapter are either sesquiterpenes or diterpenes.

Figure 6-1 - Isopreae unit and a terpene

6.2. Determination a Structures of Two Sesauiterwnes from Carrraria

The IWO compounds were collecied and provided by Dr. Paul Reese of the

University of West Indies, Mona campus in Kingston, Jamaica. They are isolated from

the plant Capraria biflora, a member of the Scrophulariaceae family. The common name

for this vascular plant is Goatweed. These plants are found in south Florida, the Keys

and tropical Arnerica. The traditional medicinal uses for extracts of this plant include

treatrnent for promoting labor, earache, indigestion, dianhea, hemorrhoids, cough, fever,

post-childbirth recover, painful menstruation, for swollen body parts, vaginal douche,

tiredness, rheumatism and kidney problems.49

6.2.1. Structure a

Frontal View

Side View

Figure 6 3 - Structure and Side View of 30

The "c, 'H and HMBC assignments for structure 30 are provided in Table 6-2.

Table 6-1 - 500 MHz 'k, 'H and HMBC data for JO

POS 1 3 ~ a 'H HlMBC

H, HO 1 143.90 (C) 7P, 6% 6P, 2 2 1 14.32 (CH) 6.3 1 (3.1,S.O) 6~~ 6P 3 90.31(C) 7P, 6P, 4a, 4P, 2 4 45.16 (CH2) 1.65 (1 1.5, 11.5, 1.4)' 2.09 (1 1.6,6.8,3.0)~ 7P, 6P, 5 5 30.52 (CH) 2.10 (m) 6a, 6P, 4a 6 38.37 (CH2) 2.19 (17.3,9.5,3.1)' 2.74 (17.3,7.3,2.0,2.0)~ 2,5 7 44.51 (CH2) 2.45 (12.5,8.3) 1.85 (12.5, 12.0, 1.4) 8a, 4a 8 35.59 (CH) 2.82 (m) 7a, 7P, 15 9 179.21 (C) 7c1,8c1 10 122.92 (C) 13, 12, 11 i l 110.19(CH) 6.48 (2.0,O.g) 13, 12,2 12 143.02 (CH) 7.40 (2.0,2.0) 13, 11 13 141.05 (CH) 7.46 (m) 12, 11,2 14 19.91(CH) 1 .16 (6.3) 6a, 401 15 15.07 (CH) 1.32 (6.1) 8 ~ , 7P

6.2.1.1. Structural Determination of a

'H, 13c, COSY, HSQC, W C and ROESY NMR experiments were performed

in CDC13. Electron impact mass spectral analysis of the product showed the molecular

ion at 246. The calculated high-resolution mass spectrum for Ci sH sOa was 246.1256

and was found experimentally to be 246.1267. The presence of the substituted furan

moiety attributed to the 'H NMR resonances at 8 6.48 (H-1 1, dd, J = 2.0,0.8 Hz), 6 7.40

(H- 12, dd, 2.0,2.0) and 8 7.46 (H- 13, m ). The conjugated olefîn showed a peak at 6 6.3 1

a Chernical shifts on 6 scaie in CDC13 In brackets, coupling constants in Hz Axial hydmgen Pseudo equatorial hydrogen

(H-2, dd, J =3.1, 2.0). Analysis of HMBC showed correlation's between the C-3

quartenas, carbon at 690.3 1 and the surroundhg protons 66.3 1 (H-2), 6 1.65(H-4a),

62.09(Ho4P), 62.45(H-7a) and 6 1.8S(HJp) and long-range 62.74(H-6).

The results of the ROESY experiments clearly showed n.0.e. between H-7a and

H-4a. There was no n.0.e. between H-7a and H-4P at the axial position. The

stereochemical assignment was assigned on this basis. A cross peak present between H-

5p and H-6P ~ o ~ r m e d the two protons to be on the same side. Similarly, a cross peak

between H-7ir and H-8 confirmed the H-8 proton to be on the same face in the a position.

Exarnination of n.0.e. data also showed that of the methyl at position 15 showed a cross

peak with H-7p and not H-7a, establishing the stereochemistry of this methyl group.

Exarnination of HSQC and HMBC spectra pieced together structure 30 for this

compound.

6.2.2. Structure a

Frontal View Side View

Figure 6-3 - Frontal view and Side view of structure 31

The 13c, 'H and HMBC assignments for structure 31 are provided in Table 6-2.

Table 6-2 - 500 MHz NMR "c, 'H and HMBC data for 31

Pos. I3c a 'H 4b EIMBC H a Ha

1 143.54 (C) 7a, 6% 613, 4P, 2a, 2 1 1 1.85 (CH) 6.10(7.5,2.8) 6% 613 3 89.84(C) 713,613, 4P, 2 4 47.28 (CH2) 1.88 (1 1.5, 1 1.5)' 1.98 (1 1.5, 5.8)d*C 14,7a, 6P 5 29.00(CH) 2.20 (m)' 14,7cx, 6a, 6P, 4P, 2 6 37.50 (CH2) 2.18 (17.5,8.7,2.8)' 2.75 (17.5,8.5, 14,4P,

2.51~ 7 42.87 (CH2) 1.87 (12.5, 1.08)' 2.55 (12.5,9.0)~ 15,8,4p, 8 34.32 (CH) 2.94 (m) 15,7a, 713 9 179.83 (C) 15,8,7P 10 122.64(C) 13, 12, 11 1 1 1 10.24 (CH) 6.45 (1.8,0.9) 13, 12,2 12 143.00(CH) 7.39 (1.8, 1.6) 13,11 13 140.94 (CH) 7.43 (m) 12, 11,2 14 20.55 (CH) 1.16 (6.7) 6% 15 15.69(CH) 1.32 (6.2) 8, 7a

6.2.2.1. Structural Determination of a 'H, 13c, COSY, HSQC, HMBC and ROESY NMR experiments were performed.

The mass of this produci was the same as that for 31 and the HRMS also confirmed that

the molecular formula was also identical. A 70:30 CsD6:CDC13 mixture was necessary

for this compound to resolve overlapping peaks, which were more problematic than 31.

The presence of the substituted îùran moiety attiibuted to the 'H NMR resonances at O

6.45 (H-11, dd, J = 1.8, 0.9 Hz), 6 7.39 (H-12, dd, 1.8, 1.6) and 6 7.43 (H-13,m ). The

' Chernical shifts on 6 scale in CDCb In brackets, coupling constants in Hz ' Pseudo axial hydrogen

Pseudo equatorial hydrogen ' Peaks are slightly broadened, due to m e r unresolved coupling. The COSY spec tm shows weak cross peaks between protons at 8 1.98 and 6 2.75

conjugated olefui showed a peak at 6 6.10 (H-2, dd, J =7.5, 2.8). Analysis of HMBC

showed correlation's between the C-3 quartenary carbon at 690.3 1 and the surrounding

protons at positions 56.10(H-2), 6 1.98(H-4) and 52.55(H-7).

Cross peaks present between H-5P and H-6P confirmed the two protons to be on

the same side. H-4P was established because it showed an n.0.e. with H-5P and

because of the axial-equatorial coupling it exhibited. H-7a showed an n.0.e. with the

equatorial H-4a proton and none with H-4p establishing its position. Examination of

n.0.e. data also showed that of the H-15 methyl showed a cross peak with H-7u, and not

H-7p showing this methyl group to be the inverted isomer to 30. Examination of HSQC

and HMBC spectra pieced together structure 31 for this compound.

6.2.3. Inter~retation of Data

The two products were determined to be sesquiterpenes of structures 30 and 31.

Configurations were successfûlly established with n.0.e. data. The products were found

to be identical and ody differed in configuration around position 15. The two

sesquiterpenes (C-15 terpenes) were found to be new compounds, which was confirmed

by a CAS online search .

6.3. Elucidation un known CaesalDnia bonduc root extracts.

Caesalpinia is a pantropical genus with approxirnateiy 150 species. The species,

Caesalpinia bonduc is a medicinai plant of wide distribution throughout the tropics and

s u b t r ~ ~ i c s . ~ ~ Many of the compounds present in the root extract of CaesaZpinia bonduc

are still unknown. Investigation of the root extracts of C. bonduc collected and provided

by Professor W.F. Tinto of the University of West Indies in Barbados, were carried out

and elucidation of the structures were performed using NMR rnethods. Previously in our

labs, chemicai investigations of Caesaipinia bonduc lead to the isolation of a new

reamnged cassane furanoditerpene, caesalpinin (Figure 6-4), being the first report of a 19

(4+3)-abeo-cassane diterpene. Further investigations of the root extracts lead to the

isolation of several new rearranged cassane furanoditerpenes.

Figure 6-4 - Structure of Caesalpinin

6.3.1 - Structures 32 and 33

- - - -

Figure 6-5 - Structures of compounds 32 and 33

The proton and carbon NMR assignments for structures 32 and 33 are shown in

Table 6-3. 'H, I3c, COSY, HSQC, HMBC and NOESY experiments were performed in

CDC13.

Table 6 3 - 500 MHz NMR 'H and 13c data for structures 32 and 33

Structure 32 Pos. 13c 'H 1 70.9(CH) 3.89(m)

2 39.1 (CH*) 2.53 (br d, 19.1) 2.20 (br d, 19.1)

3 127.7 (C) - 4 127.7 (C) - 5 76.6 (C) - 6 76.0 (CH) 5.38 (d, 8.3) 7 74.2 (CH) 5.63 (dd, 10.2,8.3)

Structure 33 'Y a 'H 34.6 (CHi) 1.44 (m)

1.53 (m) 18.2 (CH2) 1.5 1 (m)

1.71 (m) 38.1 (CH2) 1.14 (m)

1.71 (m) 38.9 (C) - 76.2 (C) - 72.3 (CH) 5.23 (t, 3.3) 3 1.4 (CH2) 1.5 1 (m)

2.19 (ddd, 13.4, I3.4,3.3)

a Chernical shifts on 6 scaie in CDC13 In brackets, coupling constants in Hz

Stnicture 32 Pos. 13c a 'H 8 48.1 (CH) 2.30 (dd, 1 1.9. 10.2)

33.2 (CH) 43.3 (C) 23.2 (CHÎ)

148.4 (C) 125.6 (C) 72.9 (C) 107.5 (CH) 141.9 (CH) 24.8 (CH3) 20.5 (CH3) 14.7 (CH3) 15.8 (CH3) - - 170.9(C=O) 21.8 (CH3) 171.1(C=O)

2.95 (ddd, 11.9. 11.2, 5.6)

2.80 (dd, 15.6,5.6) 2.48 (dd, 1 5.6, 1 1.2)

6.39 (d, 1.9) 7.25 (d, 1.9) 1.56 (s) 1.68 (br s) 1.74 (br s) 1 .O4 (s) 3.02 (d, 6.4) 3.44 (br s) - 2.1 O (s) -

2 1.7 (CH3) 1.99 (s)

6.3.1.1. Structural Determination of32

Structure 33 I3c a 'H 4b

30.4 (CH) 1.26 (m) 37.9 (CH) 2.34 (m) 4 1.4 (C) - 2 1.7 (CH2) 2.47 (dd, 18.0, 10.0)

2.5 1 (dd, 18.0, 8.0) 149.s (C) - 122.3 (C) - 31.1 (CH) 2.57 (dq, 6.8,5.7) 109.4 (CH) 6.19 (d, 1.7) 140.3 (CH) 7.22 (d, 1.7) 1 7.6 (CH3) 0.99 (d, 6.8) 27.6 (CH3) 0.98 (s) 25.7 (CH3) 1.25 (s) 16.5 (CH3) 1.34 (s)

The compound was isolated as a white solid with a melting point range between

163-165 OC and [alD +7.2 O . The molecular formula, C24H3208, was c o n f i e d by hi&-

resolution mass spectrornetry. The IR spectrum had absorption due to hydroxyl (3429

cm"), ester carbonyl (1735 cm") and furan (757 c d ' ) functionalities respectively. The

presence of the 1,t-disubstituted furan moiety attributed to the 'H NMR peaks at 6 6.39

(1 H, d, J = 1.9 Hz, H-15) and 6 7.25 (1 H, d, J = 1.9 Hz, H-16). The 'H NMR spectnim

also had resonance's for two olefinic methyl's at 6 1.68 (H-19) and 6 1.74 (H-19), two

acetoxy methyl's at 6 1.99 (7-OAc) and 62.10 (6-OAc) and two tertiary methyl singlets at

6 1 .O4 (H-20) and 6 1.56 (H-17). In addition, there were three oxymethine resonances at

6 3.89 (m, H-l), 6 5.38 (d, J = 8.3 Hz, H-6) and 6 (dd,+ 10.2, 8.3, Hz, H-7). The latter

two oxymethines were associated with the acetoxy groups attached to C-6 (6 76.0) and C-

7 (6 74.2) on the basis of 'H-'H COSY, HMQC and HMBC experiments. The third

oxymethine proton was assigned to C-1 since the C-20 methyl group at 6 1.04 showed

HMBC correlation's to the C-1 carbon at 6 70.9 as well as C-5 (876.6), C-9(633.2) and

C- 1 O(643 .3). In the ' H - ~ H COSY spectnun, H- 1 has cross peaks with methylene protons

at 62.53 and 62.20; these protons showed direct connectivity to C-2 (639.1) in the

HMQC spectrum. The methylene group at C-2 also had 'H-'H COSY cross peaks with

the methyl protons at 6 1.68(H-18) and 51.74(H-19), respectively, while these methyl

protons had cross peaks with each other, establishing their geminal disposition.

Stereochemistry was determined f'rorn vicinal coupling constants and NOESY cross-

peaks.

6.3.1.2 Structural Determination of 33

The molecular formula of C22H3204 was confhned by H R M S . The IR spectrum

had absorption's typical of hydroxyl (3447 cm"), carbonyl ester (1732 cm") and furan

(758 cm-') functionalities. The 'H NMR spectrum showed resonances indicating the

presence of the three tertiary methyl groups at 80.98 (H- 1 8), 5 1.25 (H-19) and 6 1.34 (H-

20); a secondary methyl group at 60.99 (H-17, d J = 6.8 Hz), and a more deshielded

acetoxyl group at 62.06 (attached at 16). The presence of the 1,2-disubstitued furan was

evident fiom resonances at 67.22 (H-16, d, J = 1.7 Hz) and 66.19 (H-15, d, J = 1.7 Hz).

An oxyrnethine was observed at 65.23 (H-6). Examination of HSQC and HMBC spectra

pieced together structure 33 for this compound.

6.3.2. Structures 34 and 34

Figure 6-6 - Structures 34 and 35

The proton and carbon NMR assignrnents for structures 34 and 35 are shown in

Table 6-4. 'H, 'k, COSY, HSQC, HMBC and NMR experiments were performed in

CDC13.

Table 6-4 - 500 MHz NMR 'H and I3c data for structures 34 and 35

1 -48 (m) 2 24.2 (CH2) 1.46 (m)

1.82 (m) 3 30.8 (CH3 1.50 (m)

1.54 (m) 4 42.7 (C) - 5 76.6 (C) - 6 26.4 (CH3 1.66 (m)

1.81 (m) 7 17.8 (CH2) 1.48 (m)

1.58 (m) 8 34.4 (CH) 1.78 (m) 9 38.1 (CH) 2.36 (m)

Structure 34 Pos. I3c a 'H 1 32.2 (CH2) 1.40 (m)

1.51 (m) 18.6 (CHz) 1.50 (m)

1.94 (m) 32.0 (CHt) 1.59 (m)

1.95 (m) 49.WC) - 77.3(C) - 27.8 (CH3 1 .83 (m)

2.37 (m) 24.7 (CH2) 1.53 (m)

1.80 (m) 34.6 (CH) 1.81 (m) 37.5 (CH) 2.21 (m)

Structure 35 I3c a 'H 32.3 (CH2) 1.46 (m)

a Chernical shifis on 6 scale in CDC13 In brackets, coupling constants in Hz

Structure 34 Pos. 13c a 'H "b 1 O 4 1.4 (C) -

22.2 (CH2)

149.8 (C) l22.8(C)

3 t .4 (CH) t 09.6 (CH) 140.6 (CH) 17.5 (CH3) 21.1 (CH3) 67.5 (CH3)

16.9 (CH3) 17 1.5(C=O)

2.36 (m) 2.48 (dd, 15.9,9.1) - - 2.60 (m) 6.18 (d, 2.4) 7.22 (d, 2.4) t .O l (d, 6.8) 1 .O0 (s) 4.05 (d, 10.7) 4.43 (d, 10.7) 1 .O2 (s) -

Structure 35 ' j ~ a 'H "b 42.0 (C) - 22.5 (CH2) 2.34 (dd, 16.7, 1 1.1)

2.50 (dd, l6.7,6.l) 149.4 (C) - 122.3 (C) -

3 1.6(CH) 2.62 (m) 109.5 (CH) 6.19 (d, 1.9) 140.3 (CH) 7.22 (d, 1.9) 17.5 (CH3) 1 .O2 (d. 6.8) 24.1 (CH3) 1.26 (s) 18 1.3 (CH3)

15.3 (CH3) 0.94 (s)

6.3.2.1. Structural Determination of 34

The molecular formula of CuHa04 was confirmed HRMS. The IR spectnim had

absorption's typical of hydroxyl (3487 cm''), carbonyl ester (1741 cm") and furan (757

cm-') functionaiities. The 'H NMR spectnun showed resonances indicating the presence

of the two tertiary methyl groups at 6 1 .O2 (H-20) 6 1 .O0 (H-18). At position 19, an

oxymethylene was present, displaying different resonances for each proton at 6 4.43 (d, J

= 10.7 Hz) and 64.05 (d, J = 10.7 Hz). The presence of the h a n ring was also indicated

with sirnilar resonances to compound 33 at 67.22 and 66.1 8.

6.3.2.2. Structural Determination of 35

The molecular formula of C20H2804 was cotlfinned HRMS. The IR spectnim had

absorption's typical of hydroxyl (3550 cm-'), carboxylic acid -OH and carbonyl (3300-

2506 cm-' and 1732 cm-' respectively) and furan (758 cm-') functionaiities. The 'H NMR

spectnim showed resonances indicating the presence of the two tertiary methyl groups at

60.94 (H-20), 6 1.26 (FI- 18) and a secondary methyl group at 6 1 .O2 (H-17, d J = 6.8 Hz).

The presence of the furan ring was indicated with resonances at 67.22 (H-16) and

66.19(H-15). Examination of the HMBC spectrum indicated that the tedary methyl

group at 6 1.26 (H-18) showed long-range correlation's to the carboxylic acid carbon at

6 18 1.3 (C-19), the quartenary carbon at 649.0 (C-4), the rnethylene carbon at 632.0 (C-

3) and the oxygenated carbon at 677.3 (C-5).

6.3.3. Structures 36and 37

Figure 6-7 - Structures 36 and 37

The proton and carbon NMR assignments for structures 36 and 37 are shown in

Table 6-5. 'H, 13c, COSY, HSQC, HMBC and NMR experiments were performed in

CDC13.

Table 6-5 - 500 MHz NMR 'H and data for structures 36 and 37

Structure 36 Pos. I3c a 'H "'

32.0 (CH2)

17.8 (CH2)

30.5 (CH2)

42.6 (C) 77.3 (C) 32.3 (CH2)

71.7 (CH)

39.5 (CH) 36.9 (CH) 40.9 (C) 22.4 (CHr)

149.0 (C) 12 1.9(C) 27.5 (CH) 109.6 (CH) 140.7 (CH) 17.2 (CH3) 2 1 .O (CH3) 67.0 (CH2)

17.2 (CH3) 1 70.7(C=O) 2 1.3 (CH3) 170.7(C=O) 2 1 .O (CH3)

1.42 (rn) 1.51 (rn) 1.49 (m) 1.51 (m) 1.48 (rn) 1.48 (m) - - 1.69 (dd, 13.8, 10.5) 2.16 (dd, 13.8,5.9) 5.28 (ddd, 10.4, 10.4,5.9)

1.93 (ddd, 11.0, 10.5,4.7) 2.55 (dd, 1 1 .O, 4.7) - 2.44 (dd, 15.1,6.3) 2.53 (dd, 15.1, 1 1 .O) - - 2.83 (dq, 7.3,4.7) 6.19 (d, 1.7) 7.25 (d, 1.7) 1 .O0 (d, 7.3) 1 .O1 (s) 4.05 (d, 10.5) 4.40 (d, 10.5) 1 .O8 (s)

2.07 (s)

2.07 (s)

Structure 37 ' 3 ~ a lu 0 u L 1

32.9 (CH3 1.96 (m)

19.4 (CH9

3 1.6 (CH2)

48.7 (C) 75.5 (C) 25.5 (CH-)

23.7 (CH2)

126.6 (C) 143.0 (C) 43.9 (C) 105.8 (CH)

153.8 (C) l25.7(C) 128.3 (C) 104.9 (CH) 144.3 (CH) 15.9 (CH3) 23.9 (CH3) 177.5 (C=O)

27.5 (CH3)

2. I O (rn) 1.69 (m) 2.09 (m) 1.76 (ddd, 12.6, 12.6,5.0) 2.02 (rn) - - 2.28 (ddd, 13.7,7.9,4.1) 2.62 (ddd, 13.7,8.1,2.7) 2.87(ddd, 17.8,8.1,4.1) 2.62 (ddd, 13.7,8.1,2.7) - - 7.35 (s)

- 6.73 (d, 2.1) 7.54 (d, 3.1) 2.39 (s) 1.34 (s) - 1.15 (s)

6.3.3.1. Structural determination of 36

The molecular formula, C24HM06, was confirmed HRMS. The IR spectrum had

absorption due to hydroxyl (3440 cm-'), ester carbonyl (1732 cm") and furan (758 cm-')

fùnctionalities respectively. The presence of the 1,2-disubstituted furan moiety attributed

to the 'H NMR peaks at &7.25(H-16) and 66. W(H-15). The two tertiary methyl groups

' Chemical shih on 6 scale in CDClt

69

at C- 18 and C-20 showed resonances at 6 1 .O1 and 6 1 .O8 respectively, the secondary

methyl at C-17 peak was at 6 1 .O0 (d, J = 7.3 Hz) and the two acetoxyl groups attached to

Cd and C-7 showed one peak at 62.07 (6H). The oxymethine proton had a signal at

65.28 (H-7, ddd, J = 10.4, 10.4, 5.9 Hz) and the oxymethylene group at C-19 showed

two peaks at 64.40 and 64.05(5 = 1 0.5 Hz). The secondary methyl at 6 1 .O0 (H- 1 7) was

assigned as attached to C-14 by HMBC, because it showed long-range correlation's to

carbons at 6 12 1.9(C-13) and 639.5 (C-8). In the 'H-~H COSY spectrum the oxymethine

proton at 5.28 had cross peaks to H-8 and C-6 methylene protons at 62.16 (dd, J = 13 -8,

5.9 Hz) and 6 1.69 (dd, J = 13.8, 10.5 Hz), thus supporting its assignment at (2-7.

6.3.3.2. Structural determination of 37

The molecular formula, C2 H2604, was confirmed by high-resolution mass

spectrometry. The IR s p e c t m had absorption due to hydroxyl (3565 cmd1), ester

carbonyl (1724 cm-') and furan (772 cm") functiondities respectively. The presence of

the 1,2-disubstituted h a n moiety attributed to the 'H NMR peaks at 67.54(H-16) and

66.73(H-15). The deshielded proton in the aromatic region at ti7.35(H-1 l), dong with

an aromatic methyl group at 62.39(H-17), confirmed the presence of a trisubstituted

benzofuran moiety. The I3c NMR spectnim had a resonance at 675.5 due to the

presence of an oxygenated quartenary carbon. Position 11 was confirmed when HSQC

showed the carbon attached the aromatic proton (67.35) was 6 105.8 and this, in turn,

showed correlation's to the aromatic carbons at 6 153.8(C- 12), 6 125.7(C- 13), 6 l26.6(C-

8) and long-range with the quartenary carbon at 643.9(C- 1 0).

' In brackets, coupling constants in Hz

70

6.3.4. Structures 38 and 39

Figure 6-8 - Structures of 38 and 39.

The proton and carbon NMR assignments for structures compounds 38 and 39 are

shown in Table 66. 'H, "c, COSY, HSQC, HMBC and NMR experiments were

performed in CDCIi.

Table 6-6 - 500 MHz NMR 'H and "C data for structures 38 and 39

Structure 38 Pos. '32 a 'H 4b 1 32.2 (CH2) 1.4 1 (m)

1.50 (m) 2 18.7 (CH?) 1.49 (m)

1.89 (m) 3 32.0 (CHI) 1.57 (m)

1.95 (m) 4 49.0 (C) - 5 76.8 (C) - 6 27.7 (CH2) 1.85 (m)

2.24 (m) 7 20.0 (CH) 1.68 (m)

1.8 1 (m) 8 36.qCH) 1.96 (m) 9 40.6 (CH) 2.23 (m) 10 42.0 (C) -

Structure 39 1 3 r a lu 4b

36.2 (CH2) 1.71 (m) 1.68 (m)

23.1 (CH2) 1.65 (m) 1.82 (m)

73.9 (CH) 4.76 (dd, 1 1.9,4.8)

40.4 (C) - 43.8 (CH) 1.49 (m) 26.6 (CH2) 1.48 (m)

1.89 (m) 75.7 (CH) 4.99 (m)

46.6 (CU) 2.02 (m) 46.0 (CH) 1.26 (m) 36.5 (C) -

" Chernical shifts on b scale in CDC13 In brackets, coupling constants in Hz

Structure 38 Pos. 13c a 'H 1 1 22.3 (CH2) 2.38 (dd, 14.8,8.2)

2.48 (dd, 14.8,6.8) 12 151.0(C) - 13 122.1(C) - 14 77.3 (C) - 15 107.7 (CH) 6.28 (d, 1.8) 16 140.9 (CH) 7.23(d, 1.8)

17 25.5 (CH3) 1.28 (s) 18 23.9 (CH3) 1.20 (s) 19 177.3(C=O) -

20 27.5 (CH3) 0.86 (s) 2 1 57.5 (CH2) 3.14 (dq, 15.0, 7.0)

3.29 (dq, 1 5.0, 7.0) 22 16.1 (CH,) 1.14 (t, 7.0) 3-AC

Structure 39 13c a 'H 4b 24.5 (CHZ) 2.03 (m)

2.09 (m) 122.4 (CH) 5.79 (t, 4.8) 14 1.1(C) - 73.7 (C) - 134.7 (CH) 6.40 (dd, 17.9, 1 1.9) 114.2 (CH2) "5.09 (dd, 1 1.9,2.3)

b5.45 (dd, 17.9,2.3) 23.8 (CH3) 1 -29 (s) 13.6 (CH3) 0.88 (s) 64.9 (CH2) 3.68 (d, 12.7)

3.82 (d, 12.7) 14.8 (CH3) 0.99 (s)

169.4 (C=O) - 20.9 (CH3) 2.12 (s) 170.6 (C=O) - 2 1.2 (CH3) 2.03 (s) 17 1 .O (C=O) - 2 1.6 (CH3) 2.09 (s)

6.3.4.1. Structural determination of 38

The molecular formula, CuH340s, was confirmed by high-resolution mass

spectrometry. The IR spectrum had absorption's characteristic of hydroxyl, (3528 cm"),

carbonyl ester (1718 cm"), and furan (756 cm-') functionalities. The 'H NMR spectrum

has resonances for three tertiary methyl groups at 60.86 (H-20), 6 1.20 (H-18), and 6 1.28

(H- 1 7) and a methyl ester at 63.69. The presence of the ethoxy group was evident frorn

1 H NMR resonances at 63.14 (dq, 15.0, 7.0), 63.29 (dq, 15.0, 7.0) and 61.14 (1, 7.0).

The tertiary methyl group at 61.20(H-18) had HMBC correlation's to the

methoxycarbonyl at 6 177.3 (C- lg), the methylene carbon at 632.0 (C-3), the quartenary

carbon at 677.3(C-4) and the oxygenated carbon at 676.8 (C-5).

6.3.4.1. Structural determination of 39

The molecular formula, C&13807, was confirmed by high-resolution mass

spectrometry. The 'H NMR spectrum showed resonances indicating the presence of the

three tertiary methyl groups at 80.88 (H-18), 60.99 (H-20) and 6 1.29 (H-17) and three

acetoxyl methyl groups at 62.03 (H-6Ac), 62.09 (Fi-7Ac) and 62.12 (H-3Ac). For this

compound, the presence of the k a n ring was not indicated. Instead, signals for olefinic

protons typical of a vinyl moiety were observed at 56.40 (H-14, dd, J = 17.9, 1 1.9) 65.45

(H-16b, dd, J = 17.9,2.3 Hz) and 65.09 (H-16% dd, J = 11.9,2.3 Hz).

6.3.5. Interpretation of Data

Compound 32 was detennined to be a new product and labeled caesalpinin B.^'

Caesalpinin B (32) represents only the second example of a cassane hanoditerpene with

a rearranged skeleton.'* Compound 33 was found to be the previously reported

caesaldekarin A." The other minor cassane diterpenoids, compounds 34-39, have not

been previously reported. Compound 36 was found to be a benzofuran, and it is known

that cassane fùranoditerpenes bearing a C-14 hydroxyl group cm be transformed into

benzohirans by the action of mild acid treatmerd4 It was presumed, however, that 36

was a natural product because no acid was used in its isolation process. Compounds 34,

35, 36, 37, 38, and 39 are known as Caesaldekarin H, Demethylcaesaldekarin C,

Caesaldekarh 1, Caesaideka~ J, Caesaldekarin K, Caesaldekarin J, Caesaldekarin K and

Caesaldekarin L re~pec t ive l~ .~~

6.4. Structural elucidation of Axinellamines A and B

A number of compounds with interesting biological properties have been isolated

fiom the marine sponges of the genus Axinella belonging to the class ~ e r n o s ~ o n ~ i a e . ~ ~

Many of these compounds contain nitrogen and have been found to possess cytotoxic and

antheoplastic a ~ t i v i t ~ . ~ ~ * ~ ~ TWO pyrrole alkaloid compounds were isolated fiom

Caribbean Axinella sp. and provided by Professor W.F. Tinto of the University of the

West Indies in Barbados, and their structures were examined by NMR spectroscopy

methods. The sponge was collected around Nelson Island, of Trinidad's Western

peninsula in July 1996 and identified by Mr. Richard Hubbard, Institute of Marine

Affairs, Trinidad and Tobago.

6.4.1 - Structures 40 and 41

Figure 6-9 - Stuctures 40 and 41

'H , "c, COSY, HSQC, and HMBC experiments were performed. The proton and

carbon NMR assignments for structures 40 and 41 are shown in Table 6-7.

Table 6-7 - 500 MHz NMR and 'H data for 40 and 41 -- - -

40 Position I3c a 'H 4b

- 130.1 (C) 105.6 (CH) 108.4 (CH) 1 16.7 (CH) 3 1.1 (CH2) 128.3 (CH) 132.3 (CH) 128.0 (CH) 139.8 (CH) 38.4 (CH) 29.7 (CH2) 1 1.8 (CH3) 20.1 (CH3) - -

7.98 (br s) - 5.95 (m) 6.14 (m) 6.68 (m) 3.40 (br d, 7.2) 5.68 (dt, 15.0, 7.5) 6.09 (dd, 15.0, 10.4) 6.00 (dd, 15.2, 10.4) 5.52 (dd, 15.2, 7.5) 2.05 (m) 1.32 (m) 0.86 (t, 7.0) 0.95 (d, 6.9) - -

- 7.51 (br s) 129.5(C) - 104.9(CH) 5.81(m) 103.6(CH) 5.94(m) 138.1 (C) - 3 1.1 (CH3 3 -28 (br d, 7.0) 128.2 (CH) 5.62 (dt, 15.0, 7.0) 132.0 (CH) 6.05 (dd, 15.0,9.8) 128.0 (CH) 6.00 (dd, 14.3,9.8) 139.5 (CH) 5.48 (dd, 14.3, 7.1) 38.5 (CH) 2.05 (m) 29.7 (CH2) 1.33 (m) 1 1.8 (CH3) 0.85 (t, 7.2) 20.1 (CH3) 0.98 (d, 6.8) 35.4 (CH3) - 29.3 (CH3) 1.61 (s)

6.4.1.1. - Structural elucidatioa of 40

The product was isolated as a pale yellow gurn with [alD 45.4". The molecular

formula of 40, Ci3HisN was connmied by hi&-resolution mass spectrornetry with an

experimental mass of 1 89.1525 and calculated mass of 189.1 5 18. The IR spectrum had

absorptions at 3391 cm-' due to the amine functionality and at 1662 cm-' from C=C

stretches. The 'H-NMR resonances for the two-methyl groups occurred at 60.86(H-13)

and 60.95(H-14). The peaks at 65.95, 66.14 and 66.68 are attributed to the 2-substituted

a Chernical shifts on 8 scale in CDC13 In brackets, coupling constants in Hz; m = unresolved multiplet

pyrrole. The resonances at 65.52 (H-10, dd, J = 15.2, 7.5 Hz), 65.68 (H-7, dt, J = 15.0,

7.2 Hz), 66.0O(H-9, dd, J = 15.2, 10.4 Hz) and d 6.09(H-8, dd, J = 15.0, 10.4 Hz) gave

evidence of the presence of a 1,4-disubstituted butadiene system. The stereochemistry of

the double bonds was detemined to be E due to the sizes of the vicinal 'H-'H coupling

constants. The structure of the 6-methyl-2,4-octadienyl side chah was established by

analysis of a *H-'H COSY spectrum, while the proton-carbon correlation's were

established by an HMQC expenment.

6.4.1.2. - Structural elucidation of 41

The product was isolated as a pale yellow gum and [alD -8.9'. The IR spectrum

had absorption's at 3401 cm" due to the amine fùnctionality and a stretch at 1652 cm-'

fiom C=C olefin stretch. The molecular formula of 41, C2~h2N2 was codirrned by high-

resolution mass spectrometry with an expeiimental mass of 41 8.3329 and calculated mass

of 4 1 8.3348. The molecular formula suggested that 40 was a dimerized form of 41, with

an extra three-carbon unit. Further evidence for this was provided by the 13c spectnun,

which only showed resonances for fifieen carbon atoms, suggesting a symmetrical dimer

similar to structure 3 (Chapter 2). The 'H-NMR spectrum had resonances due to the

presence of six methyl groups showing only three resonance peaks because ail were

chernicaily equivalent. Two occurred as singlets at 6 1.6 1 (H-l6/17), two occurred as a

triplet at 60.85 (H-13, J = 7.2 Hz) while the other two occurred as a doublet at 60.98 (H-

14, J = 6.8 Hz). The 2,5dibubstituted pyrrole had resonances at 65.8 1(H-3) and 65.94

(H-4).

6.4.2 - Interpretation of Data

Both products were detemined to be new pyrrole alkaloids named Axinellamines

A and B.'^ Axinellamine A (40) was detennined to be 2-((EE)-6-methyl-2,4-

octadieny1)pyrrole. Axinellamine B (41) was conclusively detemined to be a related

dimer of Axinellamine A (40), but containing an additional (CH3)2C unit between the C-

5 and C-5' carbons of the two pyrrole rings.

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