Determination of relative and absolute configuration in the annonaceous acetogenins

Determination of relative and absolute configuration in the annonaceous acetogenins

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 17 © 1995 Elsevier Science B.V. All rights reserved. 251 Determination of Relative ...

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 17 © 1995 Elsevier Science B.V. All rights reserved.

251

Determination of Relative and Absolute Configuration in the Annonaceous Acetogenins Elizabeth A. Ramirez and Thomas R. Hoye

I. INTRODUCTION The Annonaceae is a large group of trees and shrubs, found mostly in tropical and subtropical regions. There Is wide botanical diversity within this class, including 120 genera and over 2000 species. Although these plants have long been used in various folk remedies, It is only recently that the chemical source of these medicinal properties has been investigated. A wide variety of natural products has been isolated from the Annonaceae.^ However, the report in 1982 of the Isolation of the acetogenin uvaricin (1) from Uvaria accuminata^ was the first example of what is now a large and growing series of acetogenins found in these sources. More than one-hundred examples have been reported in the intervening decade.3.4 That these compounds often exhibit remarkably potent cytotoxic and other biological activity has fueled interest in this class.

2 (Qigantecin)

Figure 1. Examples of the Tetrahydrofuranyl Structure in Annonaceous Acetogenins: an Adjacent bis-THF [Uvaricin (1)], a non-Adjacent bis-THF [Gigantecin (2)], and a mono-THF [Annonacin (3)].

252 The Annonaceous acetogenins have some common structural features. Most of the compounds reported so far contain one or two tetrahydrofuran (THF) rings situated along a long, unbranched aliphatic chain. The only reported exceptions to this structure, molecules containing appropriately located epoxides and/or alkenes, can be construed as biogenetic precursors to the THF backbone."* One additional anomaly, muricatacin.s can be envisioned as arising by oxidative cleavage of monotetrahydrofuran acetogenins and has been suggested as a product of plant catabolism.6 Of those acetogenins containing two THF rings, these rings can be either adjacent (i.e., directly linked, cf. 1) or separated by a four-carbon chain [cf., gigantecin (2)]7 (Figure 1). Also, one end of the aliphatic chain is invariably terminated by a ylactone, either a,p-unsaturated [cf. 1- 3, annonin I (4),8 and bullatacin (5)^] or saturated with a hydroxyl group at 0(33) as in itrabin (7)^^ In some cases, this functionality is rearranged to an acetonylbutanolide with an oxygen at 0(4) [cf., bullatacinone (6),^ Figure 2]. Hydroxyl groups are nearly always found adjacent to the THF rings; various other hydroxyls may be scattered along the carbon chain, with the most common location at 0(4) (cf., 5), two carbons removed from the lactone moiety.

Figure 2. Examples of Various Lactone Moieties in Annonaceous Acetogenins: an a, p-Unsaturated-y-lactone [Annonin I (4)], a 0(4)-Hydroxylated a, pUnsaturated-y-lactone [Bullatacin (5)], a Rearranged Acetonyl Butanolide [Bullatacinone (6)], and a Saturated p-Hydroxy-y-lactone [Itrabin (7)].

253 The constitutional stmctural features just described were deduced by classical methods. Both ""H and '•^C NMR spectroscopy have proven valuable in unraveling these complex structures, and Interpretation of mass spectrometric fragmentation patterns is often used to pinpoint the location of substituents along the carbon chains. In recent years, two-dimensional NMR techniques have provided even more structural detail, and spectral trends among the many known compounds has made determination of connectivity a relatively straightforward task."* Determination of the stereochemical features within this new class of molecules, however, is a much less simple matter. Their waxy, amorphous, or microcrystalline nature has so far made them unsuitable for direct X-ray diffraction studies. However, given the biological properties of the Annonaceous acetogenins and their analogs, their relative and absolute configuration is an important issue. Several members of this class, possessing the same connectivity but all diastereomeric in nature, have been found to have significantly different bioactivltles,^.^ indicating that their mode of action is configuration dependent. Currently many generally applicable methods for determining configuration are available, and the three-dimensional structure of these molecules is no longer a complete mystery. Before these methods were available, however, structure determination resulted in the assignment of only a handful of stereochemical attributes In a few individual compounds.

HQ^

2)H202

OH S-Lactic Acid

Figure 3. Degradation of Uvaricin for Determination of Configuration at C(36). The first stereochemical feature reported for the Annonaceous acetogenins was the absolute configuration of C(36) in uvaricin (1 ).'•'' Uvaricin was subjected to degradation by ozonolysis with an oxidative workup (Figure 3), yielding, among other products, lactic acid. The mixture, after derivatlzation with CF3CH2N=C=0, was subjected to gas chromatography on a chiral column, and the degradation product was thus determined to be S-lactic acid. It therefore follows that C(36), from which this stereogenic center derived, is also S. Although the acetogenins presumably arise by similar blosynthetic pathways, In the absence of similar degradation experiments [or alternative methods for

254 deducing C(36) configuration] it is dangerous to conclude that all acetogenins share this configuration.3 Although, as previously mentioned, the Annonaceous acetogenins have thus far proven largely unsuitable for X-ray crystallographic studies, stmctures have been reported for two acetogenin derivatives. Pettit et al.12 conducted X-ray crystallographic studies on the 15-p-bromophenylurethane derivative 9 of rolliniastatin I (8). Later, Born et al.6 converted annonin I (4) to the saturated, crystalline potassium salt 10 (Figure 4) and successfully determined Its X-ray structure, providing the connectivity and relative configuration of this molecule. Although to date no other Annonaceous acetogenins have been similarly studied, the results of these two experiments have proven indispensable for validation of other methods for deducing stereochemical features. Neither of these studies provided the absolute configuration of the acetogenin.

O,

OK*

HO 15

MS

b 4 (Annonin I)

/. H2> Pd/C

'

2. KOH. CH^H

b

b b

OH 24

[24

HO

Figure 4. Two Acetogenin Derivatives, 9 and 10, with Single Crystal, X-ray Structures.

With an ever-increasing number of Annonaceous acetogenins being reported, there have been many efforts to develop methods for elucidation of configuration that are more generally applicable. Table 1 shows a time-line chronicling the major contributions that address various stereochemical issues in these molecules. Beginning with our report in 1986 describing the relative configuration of the bis-THF portion of uvaricin,i3 research has yielded many complementary methods for

255

determining configuration within these natural products. This chapter presents an overview of these methods (through 1993). The classification/labeling scheme for the structural subunits of the Annonaceous acetogenins proposed^ and later expanded^ by McLaughlin will be used in this chapter. 1"* The terminal lactone moiety is labeled "A", and this subunit is designated A1, A2, A3, A4, or A5 depending on Its structure (Figure 5). For example, the a,punsaturated lactone without other functionality on the carbon chain, such as found in uvaricin (1), is known as A1. The letter "B" refers to the THF subunit, labeled B1, 82, 83, or 84 ; for example, 83 refers to the non adjacent bis-tetrahydrofuranyl subunit. Other, miscellaneous functionality is represented by the letter "C": hydroxyl (C1), carbonyl (C2), acetate ester (C3), vicinal diol (C4), epoxide (C5), and olefin (C6). This scheme for organization and labeling of the acetogenins simplifies reference to molecules possessing common structural features; for example, instead of referring to "a monoTHF subunit bearing only one adjacent hydroxyl group", one can simply say "a 84 subunit." o Vo Rx.--V..>**,>/^

o OH V - o R^sX^s.^'SV^

i^ o—f o "vX^'^AsX^

o V-o n^^X-Ss..A^

o V-o ^^.y^-^^y'^Kf^

OH

A1

OH

A2

^—'

B1

A3

OH

OH

OH

A4

OH

OH

B2

B3

^—'

A5

OH

84

OH

Y

^r >" v^

Figure 5- Lactone (A#), Tetrahydrofuran (8#), and Miscellaneous (C#) Substructural Units Found in the Annonaceous Acetogenins.^

The strategy we have used to develop general methods for the determination of both relative and absolute configuration within various substructural units of the acetogenins consists of four parts, i) An appropriate set of model compounds is identified and synthesized so as to provide a complete set of diastereomers of unambiguously known

256 Table 1 . Time-line of Important Events, Including Major Advances in Determination 1982

First Annonaceous acetogenin, uvaricin, reported.

Cole et al. J. Org. Chem. 47,3151.

1985

C(36) of uvaricin determined to be S by degradation to lactic acid.

Cole et al. J. Nat. Prod. 48, 644.

1986

Model compounds of bis-THF structure prepared for NMR correlation studies; observation that ^H NMR shifts of acetate methyl groups correlate with relative configuration of C(15)/C(16) and C(23)/C(24) in uvaricin (later proven to be correct).

Hoye et al. Tetrahedron 42, 2855.

1987

""H NMR correlation studies suggest that bis-THF portion of uvaricin possesses threo/trans/threo/trans/erythro relative configuration among C{15)-C(24). Establishes general method for determining relative configuration.

Hoye et al. J. Am. Chem. Soc.

1987

Rolliniastatin I reported, and successful X-ray studies on p-bromophenylurethane derivative establish complete relative configuration.

Pettit et al. Can. J. Chem. 65, 1433.

1988

''H NMR correlation method further validated by comparison of rolliniastatin I NMR data with the now known relative configuration. Method made more quantitative, relying less on visual inspection. Relative configuration of bis-THF moiety of aslmicin verified.

Hoye et al. J. Org. Chem. 53, 5578.

1989

Sneden et al. Rollinicin reported, containing a vicinal diol along one carbon chain; relative configuration assigned as J. Nat. Prod. 52, 822. erythro based on cims fragmentation pattern.

1989

Bullatacin and bullatacinone reported; absolute configuration at C(4) assigned as S based on ORD spectral data (later proven to be incorrect); bullatacin successfully converted to bullatacinone, proving that they possess the same relative configurations along the adjacent bis-THF backbone.

McLaughlin et al. J. Nat. Prod 52, 463.

1990

Annonin I (squamocin) reported; successfully studied by X-ray analysis of a derivative. Previously described NMR correlation method gives results in accordance with structure determined by X-ray. Complementary 1H as well as i^C NMR correlation method developed to determine the configurational relationship between a THF ring and an adjacent hydroxylated carbon.

Born et al. Planta Med. 56,312.

109, 4402.

257

of Configuration, in the Development of Annonaceous Acetogenin Chemistry. 1991

"^H NMR-based method for assigning relative configuration (i.e., cis vs. trans) of 2-acetonyl-4alkylbutanolides.

Hoye et al. J. Org. Chem. 56, 5092.

1991

Synthesis of 15,16,19,20,23,24-^exep/-uvaricin (a diastereomer of the natural product) confirms relative configuration and establishes absolute configuration (via Mosher esters) of the first acetogenin, uvaricin.

Hoye et al. J. Am. Chem. 113,9369.

1992

Gigantetronenin and gigantrionenin reported, first Annonaceous acetogenins found to contain a double bond along one of the aliphatic chains. Configuration in both determined to be cis from ^H NMR coupling constants.

McLaughlin et al. J. Nat Prod. 55, 1655.

1992

Relative configuration of some mono-THF acetogenins confirmed by correlation of ^H and "^^C NMR chemical shifts with two mono-THF model compounds of known configuration.

Figaddre et al. Tetrahedron Lett. 33, 5749.

1992

Absolute configuration of C(4) in C(4)-hydroxylated compounds determined by NMR analysis of Mosher ester derivatives and comparison to model compounds. All configurations studied determined to be R at C(4). Method also applicable for determining the relative configuration between C(4) and C(36), as well as the absolute configurations of carbinol centers adjacent to THF rings.

McLaughlin, Hoye, et al. J. Am. Chem. Soc. f 74, 10203.

1993

General method for determining relative configuration of mono-THF acetogenins by correlation of 1H chemical shifts with mesitoylated model compounds.

Cassady et ai. Tetrahedron Lett. 34, 5847/5851

1993

Total synthesis of enf-bullatacin, the enantiomer of the bis-THF natural product, confirms absolute configuration of bullatacin.

Hoye et al. Tetrahedron Lett. 34, 5043.

1993

Total synthesis of solamin and reticulatacin, two mono-THF acetogenins, confirms their absolute configuration.

Keinan et al. J. Am. Chem. Soc.

115,4891.

Soc.

258 relative and/or absolute configuration, ii) An appropriate battery of spectral data from this set is carefully collected, interpreted, and tabulated, iii) Trends In these data are observed, iv) Relevant data from the natural products themselves, or appropriate derivatives, are collected and compared with those from the set of model compounds to deduce the relevant configurational relationships. Many of the general methods described herein make use of comparisons of NMR chemical shift data between a molecule with an intact natural product skeleton and another, skeletally simpler, model compound. It is more convenient to draw parallels between the two if the numbering scheme used to refer to the atoms involved are the same in both stmctures. Therefore, wherever possible, atoms on the carbon skeleton of the model compound(s) will be numbered corresponding to the natural product(s) they are intended to mimic, regardless of the "proper" numbering for the model structure.

II. THF BACKBONE 1. Adjacent bis-THF Structures (B1) In 1986, this laboratory''^ described the synthesis of a series of twelve acetylated model compounds 11a-l (Figure 6) for the bis-THF structure of uvaricin (1), one of only a handful of Annonaceous acetogenins known at the time. Among other things, we noted that the ''H NMR chemical shifts of the acetate methyl groups on the models showed a clear correlation with the relative configuration (either three or erythro) between the carbon bearing the acetoxy group and the adjacent carbon in the THF ring. Specifically, an erythro relationship between C(15)/C(16) or C(23)/C(24) in the model compounds led to a 5 of 2.051 ± 0.007 ppm, while a three relationship placed the methyl group at 2.075 ± 0.008 ppm. Since the acetate methyl group in uvaricin (1) resonates at 2.049 ppm, while the acetate derivative of uvaricin (the diacetate 12) showed acetate signals at 2.049 and 2.074 ppm, we concluded that the relationship between C(23)/C(24) in uvaricin is erythro, while the C(15)/C(16) relationship is three (Figure 7). Notice that very small differences in chemical shift were meaningful in the trend just described--the acetate methyl groups for each set of six diastereomeric compounds having either both erythro or both three terminal diastereomeric relationships all fell within a range of just over one one-hundredth of a ppm. Moreover, the two different diastereomeric environments led to a difference of only slightly more than two onehundredths of a ppm (i.e., A5 = 0.024). This requires a certain degree of care in measuring and reporting chemical shift data. To ensure reproducibility and confidence in our measured 8 values, we always include TMS as an internal standard in our samples, and we always set the TMS resonance to 5 = 0.00 ppm before printing spectral peak positions. Although this may seem obvious, it Is apparent to us that many

259

< ^Me

AcO,.,^ n-CsH,^

AcO,,,^ n-CgH^^ AcO,

fl5 ^16 O

erythrocis

n-CsH,, AcO,,.^ n-CsHn

P

I P

AcO,,.^n-C5Hii

I P

"MQ

threo-

J 20

cis

>

123 l24

erythro-

"^Mo

AcO**'^n-C5Hii

AcO^^^n-CsHn AoO^''^n-CsHn AcO^^nCgHt,

AcO*'*^n-CgHi,

\AB

11a

lib

11c

erythro/cis/threo/cis/erythro « er/c/lh/c/er

erlVth/der

er/t/th/t/er

lid er/c/er/c/er

lie erA/er/c/er

111 erfV&rfVer

AcO^^n-CsHii

AcO^^n-CsHii AcO^^n-CsHn

AcO^^n-Crf-lii

AcO^^n-CsHn

AcO^^n-CsHn

AcO*

AcO^^n-CgHt^ AcO*'

AcO

AcO**

AoO

n-CgHii

n-CsHn

n-C^n

iig

11h

111

Ih/c/th/c/th

th/VtfVc/th

iy

tlWtfWth

th/c/er/cmi

n-CsHn

tt/t th/Ver/c/th

n-CsHn

111 fhn/erfm

Figure 6- Twelve (of Twenty Possible) Diastereomeric, Synthetic, Model Compounds for the bis-THF Core of B1 Acetogenins. researchers are in the habit of referencing spectral resonances to some standard value of the solvent peak (e.g., residual CUCb in the CDCI3 to 8 = 7.26). This is dangerous because the solvent chemical shift is solute-dependent (e.g., CHCI3 is a weak hydrogen-bond donor); the inert TMS standard much less so.

or both down (i.e.. threo)

r^]"^

O

HgC^ O S 2.049

5 2.049

Figure 7. Acetate CJbis Chemical Shifts of Uvaricin Acetate (12) Define the C(15)/C(16) and C(23)/C(24) Relative Configurations for Uvaricin (1).

260 Further comparison of the model compounds H a - P ^ with uvaricin acetate (12) supported the assignment of C(23)/C(24)-erythro and C(15)/C(16)-threo. This analysis contributed additional information about the three-dimensional structure of uvaricin (1). Each stereorelationship along the THF backbone (three and erythro for pairs of adjacent oxygenated carbons, or cis and trans across THF rings) was correlated with a distinctive set of chemical shifts for the protons along the bis-tetrahydrofuranyl structure. The differences observed were in some cases small, but still significant enough to impart confidence In deducing the relative configuration along the B1 subunit. The chemical shift patterns of the twelve model compounds and of uvaricin and uvaricin acetate are recorded in Table 2 and shown graphically in Figure 8. (Due to the symmetry of the model compounds, and to simplify the graphic, we here revert to the numbering scheme used in the original paper.) In addition to the previously noted acetate methyl shifts, key observations were: i) H(5) and H(2) each appeared 0.04 - 0.08 ppm downfield in the trans/trans models compared to the cis/cis Isomers (where cis and trans refer to the substitution pattern on each THF ring); ii) if the configuration of the model is either cis/cis or trans/trans, H(2) is shifted farther downfield for the relationship C(2)/C(2') = three than for C(2)/C(2') = erythro; and iii) in the unsymmetrical (cis/trans) models, the resonances for H(2) and H(2') are nearly superimposed for C(2)/C(2') = erythro but significantly separated when C(2)/C(2') = three. Visual comparison of the ''H NMR spectrum of uvaricin acetate (12) to the model compounds yielded the closest match with the .../trans/threo/trans/... models; this, coupled with the previous information about the C(15)/C(16) and C(24)/C(25) relationships led to the conclusion that uvaricin has a three/trans/threo/trans/erythro configuration along the THF backbone, proceeding from carbon 15 to carbon 25 (see Figure 8). Table 2.

^H NMR Chemical Shift Values (in ppm) for the Methine Protons Associated with Oxygenated Carbons In the Model Compounds l l a - l .

1 ^ Configuration

H(2)

H(2')

H(5)

H(5')

H(6)

H(6')

Ac

Ac'

11a

er/c/th/c/er

3.81

3.81

3.94

3.94

4.90

4.90

2.045

2.045

lib

er/t/th/c/er

3.76

3.88

3.93

4.01

4.91

4.91

2.053

2.048

11c

er/tAh/t/er

3.88

3.88

3.98

3.98

4.91

4.91

2.045

2.045

lid

er/c/er/c/er

3.71

3.71

3.91

4.95

er/t/er/c/er

3.80 3.84

3.91

3.91 3.97

4.95

lie

4.91

4.96

2.045 2.058

2.053

3.99

3.99

4.92

4.92

2.050

2.050

3.86

3.93

3.93

4.94

4.94

2.069

2.069 2.077

2.045

lit

er/t/er/t/er

3.80 3.84

iig 11h

th/c/th/c/th

3.86

th/t/th/c/th

3.84

3.93

3.91

4.08

4.88

4.88

2.077

111

th/t/thAAh

3.90

3.97

3.97

th/c/er/c/th

3.77

3.93

3.93

4.85 4.84

4.85 4.84

2.074 2.074

11| 11k

3.90 3.77

th/t/er/c/th

3.82

3.82

4.850

th/t/er/t/th

3.84

3.84

3.93 3.97

3.97

111

3.97

4.84

2.073

2.073

4.852

2.080

2.075

4.84

2.071

2^07;^J

261

er/c/tfi/c/er er/t/th/c/er

o^Ms

er/t/th/t/er

nc f/ef

er/t/er/c/er er/t/er/t/er

-J5-J ;

th/t/th/c/th .n-CsHii t h / t / t h / l / t h

15

Figure 8.

th/t/th/l/th

I

A

2

A_..

I..5. 11k 111

: 5.0

.5._5lL...J2 5 i 2

6

4.9

r 4.8

f2 74

A

5 !

... i.. 6 !-«•

....lij... 2i^15

H(24)

A

•.A...

2415

I

...A.

i.

.A;A_.

4.1

-4-

th/c/th/c/er

.A:A.

I

5(ppm)

th/t/th/t/er

.-_.A.

J5-...J.?....:.

* __..5

..6..;. 6 f^ ....6!..

;.

i..2i.;.

^

._.j5l

..e..i :«--^

Ih/c/th/c/th

Ih/c/er/c/th th/t/er/c/th th/t/er/l/lh

t d .....^-.

rs 24/15;

.;

4.0

3.9

-4—

-4—

"T" 3.8

u.-Ji 23/16 20/19

.; ;

3.7

-I 1-—

i ...i

J.J.J AcO

'1623 i 20/19' 23/16201/19

\

AcO



i.Ul..LJ.L -.:-l-—1-

2.08 2.06 2.04

i

-^-

AcO

AcO

AcO's!

Graphical Comparison of Proton Chemical Shift Data for Model bisAcetates 11a-l and the Peracetate Derivatives of Uvaricin (12), Rolliniastatin I (14), and Asimicin (15).

In that early work w e relied much more on proton than carbon chemical shift trends. This was, in part, driven by the limited quantities of some of the twelve pure, synthetic, model compounds. However, the proton shift trends were also more meaningful than the carbon for this particular set of model compounds. It is our contention that proton chemical shift data should be used more frequently for this purpose and that this underutillzation Is largely a bias of technological origin. From the advent of '•^c NMR spectroscopy chemical shift trends were recognized to be of primary importance. Relatively large field dispersion and the routine lack of coupling data predisposed

262 researchers to rely heavily on "^^c chemical shifts. In the case of protons, only in the last decade has the routine availability of spectra recorded at Increasingly higher magnetic fields provided relatively complete assignments of the majority of resonances in spectra of complex molecules. Thus, proton chemical shift trends in complex molecules now warrant very careful attention. The 1987 report of the X-ray structure of a derivative of rolllniastatin I (8)''2 permitted us to further validate this ''H NMR correlation method."'^ The relative configuration along the THF rings in rolllniastatin I (8) (I.e., threo/cis/threo/cis/erythro) as determined by chemical shift correlation (Figure 8) matched exactly with the crystallographically determined structure. In the course of this analysis, some refinements to the method were made. Unlike uvaricin acetate (12), the triacetylated rolllniastatin I (14) did not exhibit a clear correlation with a single set of model compounds; two possibilities for the relative configuration were identified by simple visual inspection. Therefore, it was necessary to make the method more quantitative to arrive at an unambiguous conclusion. This was accomplished by comparing each of the eight measured "'H chemical shifts [H(15), H(16), H(19), H(20), H(23), H(24), and the two acetate methyl groups] for the natural product derivative with the analogous resonances for each of the model compounds, and taking the sum of the observed chemical shift differences. (For the six, unsymmetrical diastereomers of the model compounds that were JTJQI made, the expected chemical shifts were extrapolated from the relevant symmetrical model compounds.) The model having the smallest sum of the absolute values of chemical shift differences (I|A5's|) compared with the natural product represents the most likely relative configuration. The results of this comparison both for rolllniastatin I (8) and for aslmicin (13), another recently (at the time) discovered Annonaceous acetogenin,''^ are summarized In Table 3. Rolllniastatin I triacetate (14) shows the best match with a hypothetical erythro/cis/threo/cis/threo model, which corresponds exactly with the relative configuration determined by X-ray crystallography on the derivative of the natural product. Aslmicin triacetate (15) was determined to be threo/trans/threo/trans/threo, which is the same conclusion reached by visual inspection. A comment must be made at this point about the limitations of this method. It leaves open the question of directionality of the stereochemical relationships. For example, were the complete relative configuration of rolllniastatin I (8) not known from the x-ray crystallographic study, it would not be possible to tell whether the order of relative configuratlonal relationships proceeding from C(15) to C(24) was threo/cis/threo/cis/erythro or erythro/cis/threo/cisAhreo. We refer to this as the "endedness" problem, and it is an issue of structural ambiguity that has been overlooked In a number of instances.

263

Table 3.

Quantitative Comparison of ''H NMR Chemical Shifts for the Peracetates of Uvaricin (12), Rolliniastatin I (14), and Asimicin (15) with each of the Twelve Model Compounds lla-l as Well as with Eight Additional Extrapolated Unsymmetrical Isomers. 2:|A5's| model

12

14

15

er/c/th/c/er er/lAh/c/er er/l/th/t/er

tra lib 11c

0.11 0.17 0.21

0.36 0.52

er/c/er/c/er

lid

0.30 0.28 0.08 0.62

5 6 7

er/l/er/c/er er/t/er/t/er th/c/th/c/th

lie 11f

0.34

8 9 10 11

thMh/cAh th/lAh/tAh th/c/er/c/th

0.44 0.28 0.36 0.32 0.02

th/l/er/c/th

111 11k

0.15 0.19 0.19 0.23 0.29 0.27

th/l/er/t/lh

111

0.22

0.15 0.21

0.22

12 13

er/c/th/c/th

11a/11g

0.26

0.09

0.36

entry

descriptor

1 2 3 4

119 11h 111

0.18 0.26 0.32 0.12 0.44 0.28

0.41

0.16 0.72

0.38 0.16

14

er/t/th/c/th

11b/11h

0.19

0.15

0.27

15

th/t/th/c/er

11h/11b

0.35

0.26

0.41

16 17

er/t/th/l/lh

0.05 0.47

0.09 0.54

18 19

er/l/er/c/lh th/t/er/c/er

20

er/l/er/t/lh

11C/11I 11d/111 lle/11k 11k/lie 11f/111

0.22

er/c/er/c/th

0.26 0.26 0.14

0.30 0.19 0.16 0.17

0.34 0.32 0.22

Born et. al.^ have reported a complementary technique to determine the relative configuration between a carbon in a THF ring and an adjacent carbinol center. This approach is applicable to all B1, 82, B3, or B4 substructures. The model compounds 16-ef and 16-f/i (Figure 9) were synthesized as a mixture of diastereomers and separated chromatographically as their acetate derivatives. These acetates were assigned as three or erythro by the observed ^H NMR coupling constant between H(15) and H(16). The isomer with the smaller coupling constant (J15/16 = 5.2 Hz) was assigned the three relative configuration while that with the larger (J15/16 = 6.0 Hz) was assigned as erythro. Such a small difference in J's suggests that this assignment was somewhat tenuous. However, it has since been confirmed by subsequent stereospecific synthesis and correlation of model mono-THF compounds."^^^o jhe

264

i^

O I

i^-'CioHai

(^

7\

63.84(82.29)"

^\1^15^^10^21

O ""5 3.84(71.83)

6 3.79(82.47)'

^ ^ , ^ " " 5 3.40 (73.87)

Figure 9. Diagnostic Proton (and Carbon) Chemical Shift Data for Simple erythroand threo-a-HydroxyalkyI Tetrahydrofurans. acetates were then reconverted to the free alcohols 16, which were studied by ''H and "•^c NMR spectroscopy. A correlation was found between the three or erythro configuration of the models and the chemical shifts of nearby ''H and ""^c nuclei, particularly C(15) and H(16). These results are summarized in Table 4 along with the relevant data for annonin I (4), the discovery of which was reported in the same paper. It is clear from these data that annonin I possesses one three and one erythro relationship between C(15)/C(16) and C(23)/C(24). The question of which was which (I.e., the endedness), however, was resolved only through X-ray crystallographic structural analysis of the derivative 10. This work provided another verification of our original approach to assignment of bis-THF relative configuration. Table 4.

Correlation of ''H and "•^c NMR Chemical Shift Values Between Annonin I (4) and the Diastereomeric Pair of Model Compounds 16-er and 16-th. 16-er

4 (Annonin 1)

16-th

H(15)

3.84

3.40

H(15), H(24)

3.40, 3.87

H(16) C(15)

3.84 71.83

3.79 73.87

H(16), H(23) C(15). C(24)

3.88, 3.87 71.7,74.1

1 C(16)

82.29

82.47

C(16), C(23)

83.4, 82.9

All of the methods discussed so far, however useful, still leave open the question of absolute configuration. In 1992, we described a study carried out in collaboration with the McLaughlin group^"' detailing our studies of Mosher ester (i.e., methoxytrifluoromethylphenylacetate) derivatives22-24 of various carbinol centers in the acetogenins. Since all THF-contalning acetogenins have at least one hydroxyl group in the B subunit, Mosher derivatization of these groups provides an opportunity to draw conclusions about the absolute configuration in this portion of the molecule. The principle behind the Mosher ester technique is illustrated in Figure 10. The two enantiomers of the Mosher acid chloride, (f?)-MTPA-CI and (S)-MTPA-CI, are used to derivatize a stereo-

265 genie carbinol center to the (S)- and (f?)-MTPA esters, 17-Sand 17-/?, respectively.25 Assuming that the preferred conformation is as shown, with the trifiuoromethyl group eclipsed with the carbonyt, conclusions can be drawn regarding the absolute configuration of the carbinol center based on "^H and ''^F NMR spectroscopic data.26 Since the phenyl group will tend to have a shielding effect on nearby atoms, protons in the L3 portion of the ester should appear farther upfield in the "• H NMR spectrum of 17Sthan in 17-/?, while those in the L2 substituent should display the opposite trend. more highly shielded

less highly shielded OMe

^<='S less highly shielded

more highly shielded

17S

t7fl

Figure 10. Dominant Conformations of Diastereomeric Mosher (MTPA) Esters of Generic Carbinols Indicating Various Shielding Environments for Protons. To confirm the applicability of this method to the tetrahydrofuran portion of the Annonaceous acetogenins, the symmetric model bis-THF 18, where both C(15) and C(24) were known to be f?, was synthesized and converted to the (S)- and (f?)-MTPA esters, S-MTPA-18 and f?-MTPA-18.2i The chemical shift differences of selected protons in these diastereomeric esters are shown in Figure 11. As expected, the sign of

63.81

HO/, n-CgH^

53.96--.^"

\ n-CgH^,

OCH3 14C^v^Bp^^(CH2)7C^^

81.66-1.58

^AOH^HQCH^CH^TC^

-¥ 0.04 A ^(14)

18

S'MTPA'18

61.62-1.54

R'MTPA-18

Figure 1 1 . Chemical Shift Differences (A6's) for Key Protons in the MTPA Esters of the Synthetic Model f?,f?-Diol 18.

266

the chemical shift difference (A5 = 6S - 6R) is negative for H(16) and H(19) in the L^ portion of the molecule and positive for H(14) In the L^ portion. Results for hexepiuvarlcin (19), a synthetic acetogenin of known absolute configuration,^^ were similar. This study2i resulted In determination of absolute configuration of certain stereogenic centers for several acetogenins. Among them were bullatacin (5) and bullatacinone (6), where the B1 subunit of the molecule possesses "unlike ends" (i.e. three/.../.../.../erythro or erythro/.../.../.../three). The mono-MTPA derivatives were made and the position of the ester determined by mass spectrometry. This allowed both unambiguous determination of absolute configuration and a solution to the endedness problem described earlier. This work, carried out in In the course of our synthesis of /7exep/-uvaricin (19),27 represented the first instances for which the entire stereostructure of any acetogenins was deduced (Figure 12).

Figure 12. First Acetogenins for Which the Entire Stereostructure was Determined.

2. mono-THF Structures (B2) Until recently, researchers wishing to assign the relative configuration of subunit B2 relied on the NMR correlation methods developed for other subunits, as just described. For example, the mono-THF-containing annonacin (3) was assigned a three configuration between C(15)/C(16) and C(19)/C(20) by the methods of Born et al, and designated as trans across the THF ring by comparing the ''H chemical shifts of its acetate with our previously described bis-THF model compounds 11a-l.28 This threo/trans/threo assignment was later verified."'^ OH

HO,..

''^sX^^s/\/\ IS

b 20 -

3 (Annonacin)

OH

o. 'VO

.4^>ur

HO,..^ MS

L '° 1 20

Figaddre et al. reported in 199229 the first use of mono-THF model compounds containing two a-hydroxyalkyi substituents to study the stereochemical features of

267

mono-THF acetogenins. Two synthetic Intermediates, 20a and 20b, of known relative (and absolute) configuration and both possessing a trans-substituted THF ring were studied by iH and ^^c NMR spectroscopy. The chemical shift patterns in these free alcohols were compared directly with those of natural products of known relative configuration (as determined by extrapolation of the bis-THF stereostructure determination methods described above), in hopes of finding a diagnostic pattern. This pattern appeared in the i^C and "^H chemical shifts at positions 15,16,19, and 20 (Table 5). These shifts showed excellent correlation between the natural products examined, murisolin (21) and annonacin A (22), and the models possessing the same relative configuration. Figadere's model compounds can be used to distinguish acetogenins having threo/trans/threo or erythro/trans/threo structures, but only these two out of the eight possible diastereomeric relationships were modeled.

Table 5.

^H and "i^C NMR Chemical Shifts of Synthetic Intermediates 20a and 20b and Two mono-THF Acetogenins, Murisolin (21) and Annonacin A (22). H(15)| H(16)

H(19)

H(20)

C(15)|C(16)|C(19)

C(20) 1

20a 1 (threoArans/threo)

3.41

3.80

3.80

3.41

74.04

82.71

82.71

74.01

20b (erythro/trans/threo)

3.82

3.82

3.82

3.38

71.56

83.24

82.15

74.33

Murisolin {21) (threo/trans/threo)

3.38

3.76

3.76

3.38

74.2

82.7

82.7

74.2

Annonacin A (22) 1 (erythro/trans/threo)

3.82

3.82

3.82

3.40

71.62

83.32

82.31

74.361

A year later, Cassady published the synthesis^^ and study^^ of a set of model compounds representing the full spectrum of possible stereochemical relationships in dihydroxylated mono-THF acetogenins. Six^O mesitoylated compounds (23a-f, Fig. 13) were synthesized and subjected to 1H NMR spectroscopic studies. In these bismesltoates, good correlation between the relative configuration and the chemical shifts of selected protons was observed (Table 6). Specifically, It was found that: (I) where a three relationship existed between C(15) and C(16) [or C(19) and C(20)],

268

MesO^^ C 4 H g

cIUo" o

M G S O *^ C ^ H g

MesO,, ^C4H9

MesO^

C4H9

120 C4Hfl

MesO*

[20 C4H9

c/I"

MesO*

c/I"

MesO^^^C4H9 |15

MesO^'*

MesO /,.^C4Hg *fl5

120 C4H9

MesC^

[20 C4H9

MesC

120 C4H9

23a

25/7

23c

23d

23e

23f

(th/c/th)

(er/c/er)

(th/c/er)

(ttVt/th)

(er/t/er)

(th/t/er)

Me

O

0-1

MesO- s mesitoate Me'

Me

Figure 13. Six Mesitoylated Model Compounds for the B2 Subunit. H(15) [or H(20)] displayed a chemical shift of <5.37 ppm, while an erythro relationship gave rise to a 5 of >5.43 ppm, (ii) where a carbinol center is involved In a three relationship, the 2,6-methyl groups of the attached mesitoate ester will have a chemical shift of >2.45 ppm, while an erythro relationship produces a chemical shift of <2.40 ppm for these methyls, and (ill) a cis relationship across the THF ring will cause the THF ring protons, H(16) and H(19), to display a chemical shift of 3.89-3.97 ppm, while a trans relationship causes a downfield shift to 4.00-4.11 ppm. Table 6.

Important Chemical Shifts in the Cassady Model mono-THF Mesitoates 23.

Compound

Relative Configuration

6H(16). H(19)

5H(15), H(20)

5 H(Me's)

23a

threo/cls/threo

3.97

5.29

2.45

23b

erythro/cis/erythro

3.93

5.45

2.40

23c

threo/cis/erythro

3.89, 3.97

5.37, 5.43

2.39, 2.48

23d

threo/trans/threo

4.09

5.35

2.45

23e

erythro/trans/erythro

4.05

5.48

threo/trans/erythro

4.00,4.11

5.30, 5.52

[ 2 3 1

2.40

2.39.2.51 J

To apply this method to mono-THF acetogenlns, a "difference minimization" scheme similar to that earlier described''^ was used. That is, the differences in chemical shifts between a model and the natural product for all relevant hydrogens were added, and this value was compared for each of the models. The model with the smallest sum of differences is concluded to have the same relative configuration as the natural product.

269

In this case, the natural product annonacin (3) was first determined to have a symmetrical structure (I.e. not threoArans/erythro or threo/cis/erythro) by the fact that protons in analogous positions were superimposed in the 1H NMR spectrum (as in Table 6, the protons are resolved in an asymmetric structure). It then remained only to determine if the relationships In annonacin were threo/cis, erythro/cis, threo/trans, or erythro/trans. The ring methine [H(19)-H(20)] and ester methine [H(15)-H(20)] protons in annonacin per-mesitoate were compared with the model compounds 23 (Table 7). These results clearly show that annonacin (3) possesses the threo/trans/threo relative Table 7.

1

Difference Minimization Data for Identifying the Best Fit (threo/trans/threo) Between Mesitoate Models 23 and Annonacin (3).

ZA5H = |6Hannonacin - SHmodellRing Methine + ISHannonacIn " 8HmodellEster Methine Ring Methine [C(16)/C(19)]

Model SHannonacIn 8H model Configuration threo/cis

4.13

|A8H|

Ester Methine [C(15)/C(20)] |A8H|

ZASH

5.29

0.03

0.19

SHannonacin SHmodel

3.97

0.16

5.32

erythro/cis

4.13

3.93

0.20

5.32

5.45

0.13

0.33

1 threo/trans

4.13

4.09

0.04

5.32

5.35

0.03

1 erythro/trans

4.13

4.05

0.08

5.32

5.48

0.16

0.07 0.24 1

configuration. This method is also applicable to the B4 subunit, containing only one hydroxyl group a to the THF ring. Cassady's observations are consistent with similar observations we have made on the analogous acetate esters.^o

Figure 14. Acetogenins for Which the Absolute Configuration of the B2 Subunit Has Been Determined.

270 Again, these correlation methods leave the question of absolute configuration unanswered. However, the absolute configuration of the B2 unit in reticulatacin (24), isoannonacin-10-one (25), annonacin-10-one (26), and annonacin (3) has been determined by the Mosher ester method described earlier (Figure 14).2t The configuration of all carbinol centers flanking THF rings in these particular compounds was determined to be R. Given the previously determined threo/trans/threo relative configuration of all these molecules, the absolute configuration of the entire THF portion was assigned.

3. non-Adjacent bis-THF Acetogenins (B3) The several acetogenins containing the B3 or non-adjacent bis-THF substructure all have one THF ring with two adjacent hydroxyl groups, and a second ring flanked by one hydroxyl group [e.g., gigantecin (2, Figure 1)]. Given the above constitution, methods already described can be applied to each mono-THF portion of the B3 acetogenins. Indeed, the relative configuration of some nonadjacent bis-THF molecules has been proposed based on such methods.'^ However, since the two THF's are separated by only two carbons, it is possible that each subunit exerts an influence on the chemical shift patterns of the other, which could possibly perturb the data. In addition, the known stereostructure determination methods can only treat the two portions of the THF backbone as isolated entities and provide no means of determining the stereochemical relationship between the two. That is to say, there is yet no way to confidently distinguish, e.g., isomers 27a and 27b (Figure 15). It would be useful to have a method to completely and unambiguously assign the configuration of this type of structure.

27a

27b

Figure 15. Ambiguity Exists (cf., 27a vs. 27b) in the Relative Configuration Between Internal Carbinol Centers in All non-Adjacent bis-THF Acetogenins (B3).

III. TERMINAL y-LACTONE (AND ADJACENT 4.0H) As stated earlier, virtually all Annonaceous acetogenins possess a y-lactone at the terminus of the carbon chain. The known structural variations within this lactone ring ("A") are summarized in Figure 5. Although the absolute configuration at C(36) in

271 uvaricin was the first stereochemical feature to be deduced for the Annonaceous acetogenins, further stereochemical studies have focused almost exclusively on the tetrahydrofuran backbone. Attempts have been made to find a general method to determine the stereostructure of the lactone moiety. These include the use of Hudson's rule and/or optical rotary dispersion methods.9.3i-32 However, we view these approaches as tenuous since they are based upon data from less than ideal model compounds. As mentioned earlier, the absolute configuration of C(36) in uvaricin has been unambiguously determined to be S^^ Presuming a similar biosynthetic pathway for all Annonaceous acetogenins, it is tempting to assume that all such natural products possess this absolute configuration. However, this need not be the case. It is necessary either to perform a chemical degradation on each acetogenin, which may not be practical in all cases, or to develop a new method for unambiguous assignment of the lone, remote C(36) stereocenter In A1 acetogenins. Another early assumption was that the C(36)/C(4) relative configuration in 4-hydroxy butenolide-containing (A2) acetogenins could be determined by direct comparison of NMR shift data. For example, bullatacin (5) was assumed to have the same relative configuration (4S*,36f?*) determined for rolliniastatin I (8) because they exhibited "essentially the same" ^H and ^^C NMR signals for subunit A2.9 However, in the course of preparing model compounds for the structure A2, we observed that the ''H and "'^C NMR behavior of this structural unit was virtually identical regardless of the relative configuration.3'33 Clearly, similarity by NMR spectroscopy is not enough to establish the relative configuration [fT.R* (or like) or fT.S* (or unlike)] of C(36)/C(4).

1. Unsaturated Lactones Having a Hydroxyl Group at C(4) [subunit A2] Recently, general methods have been developed to assign both the absolute configuration of C(4) and the relative configuration between C(4) and C(36) in acetogenins possessing the A2 subunit.2''.34 in these acetogenins, which have a hydroxyl group at C(4), the absolute configuration of this carbinol center can be determined by the Mosher ester method described earlier in this chapter.2i Model compound 28SS, of known (S,S) configuration at C{4) and C(36), was prepared and derivatized with both enantlomers of the Mosher acid chloride. The A8H and A8F obtained for these derivatives are consistent with the known S configuration at C(4). Extension of this method to the A2 subunit in several acetogenins (Figure 16) has shown that the configuration at C(4) in all these compounds is R. Note that this result disproved a previous assumption that bullatacin (5) has the S-configuration at C(4).9

272

OH ^ V - O

28-SS

28-RS

OH

>-0

28'SR

Figure 15. Absolute Configuration of the A2 Subunit In Bullatacin (5), Asimicin (13), RoHiniastatin I (8), Annonacin (3), and Annonacin-10-one (26) Is (4H,36S) [or (4f?,34S) for 3 and 26] from Comparison of their Mosher Esters with Those of Synthetic Model Compounds 28.

Further work has enabled us to extend this method to determine the relative configuration between C(4) and C(36) in the A2 subunit .20,34 Whereas the method just described (and indeed, the conventional use of Mosher ester analysis) relied on the sign of the chemical shift differences to determine the absolute configuration, we have found that the C(4)/C(36) relative configuration is reflected in the magnitude of these differences. This phenomenon was observed for all the Annonaceous acetogenlns we studied. In addition to the model compound 28-SS described above, its stereoisomers 28-f7Sand 28-S/? were synthesized and derivatized with (R)- and (S)-MTPA-CI, respectively, and the Individual sets of chemical shift differences In their ""H and ""^F NMR spectra were compared. These A6 data, along with those from the relevant portion of bullatacin (5), are shown as absolute values graphically in Figure 16. It is clear from the graphic that the magnitudes of the chemical shift differences fall into definite patterns depending on the relative configuration between C(4) and C(36). Thus bullatacin (5),

273 along with the other Annonaceous acetogenins we studied, was determined to possess FT.S^ or unlike relative configuration between these stereogenic centers. Since all of these acetogenins had been determined to have an /? configuration at C(4) by the methods described earlier, it follows that C(36) is S. This information establishes the complete stereostructure for all five natural products studied, with the exception of C(10) In annonacin, 3 (Figure 15). • ^HData

«

» ^^hUata ||A5FI

|A8HI

(ppm)

(S)-7SS/(fl)-7SS

(S)-7S/?/(S)-7f?S

(ppm)

Bullatacin(l)

1 r

U.J

,A n +U.O

k-+0.4 U-+0.3

0.2

0.1

. -- . .

.-..._...« -.....-

0.0

j]

\lu

1—1—1—\—1—1

37 36 35 3a/b 4

Like

5

. _1 L......---.

J . . . . J U-+0.2 h-+0.1

m "Tl

1 1U—-on 1 1LLJ I l l 1—1—1—1—1—1 r"1—1—1—11—1 37 36 35 3sJb 4

Unlike

5

\37 36 35 3a/b ^

5

36S4f?

Figure 16. Graphical Representation of Absolute Values of A8H and A8F Data from Models 28 vis-a-vis Bullatacin (5): Magnitudes, Not Signs, Permit Assignment of C(4) vs. C(36) Relative Configuration. 2. Rearranged Acetonylbutanolides (A3) In 1991, we reported a method^s for assigning the relative configuration between C(2) and C(4) in the acetogenins containing an acetonylbutanolide moiety (A3). First, cis and trans model compounds 29c and 29f, respectively, were synthesized, and the relative configuration of each was unambiguously assigned through nOe studies and conformational analysis to explain the observed values of coupling constants. With these assignments in hand, the model compounds were examined by ''H NMR spectroscopy in both CDCI3 and CeDe. In both solvent systems, the two diastereomers were found to contain many characteristic differences in their chemical shift and coupling patterns (Table 8). Most diagnostic are the following: (i) H(4) occurs >0.1 ppm downfield in the trans isomer compared with the cis, (ii) the geminal hydrogens H(3a) and H(3p) are closer together (A6, of --0.2 ppm vs. ~1.1 ppm) in the spectrum of the trans compounds vs. the cis, and

274 (iii)

H(4) and H(3|3) in the trans diastereomers exhibit relatively small coupling (J = 3.9 Hz for [29f|) while in the cis isomer the large coupling (J = 9.8 Hz for [29c]) reflects their trans-diaxial-like orientation. o o—f

.o o

o' ' y 3< 6^^

29c Table 8.

29t

""H NMR Data Reflecting Diagnostic Differences in Model Diastereomeric Acetonyl Butanoiides 29c and 29t. Compound #

|

In CDCI3

c/s-Model 29c

frans-Model 29t

Isoannonacin 31

lsoannonacin-10-one 25

5H(4)

4.41

4.55

4.54

4.55

JH(33)/H(4)

A5H(3a) - H(33) ^Configuration

9.8

3.9

3.6

3.6

+ 1.09

-0.22

-0.27

- 0.24

cis

trans

trans

trans

Compound # In CeDe

c/s-Model 29c

frans-Model 29t

5H{4)

3.69

4.02

Bullatacinone Bullatacinone Squamone (Minor) (Major) 30 6b 6a 4.05

3.72

JH(33)/H(4)

9.8

3.9

A5H(3a)/H(33) 1 Configuration

+ 1.10

-0.31

-0.30

+ 1.08

cis

trans

trans

cis

4.00

trans

|

Comparison of these results with the published spectral data of acetogenins with the A3 subunit and their derivatives yields strong evidence for the relative configuration of the lactone moiety. These results (Table 8) show that the mono-THF acetogenins squamone (30), isoannonacin (31), and isoannonacin-10-one (25) possess a trans relationship between C(2) and C(4); and in the bis-THF acetogenin bullatacinone (6), which was isolated as a mixture of cis and trans diastereomers, the trans configuration predominates (Figure 17). Rollinone (32) was shown to be an --1:1 mixture of cis and trans isomers. Notice that in those structures the absolute configurations of C(15)-C(24) is known only for 6a and 25; for all other structures in Figure 17 only the relative configurations along the THF core and, independently, across the butanolide ring are known.

275

Figure 17. Acetogenins Containing the Rearranged Acetonyl Butanolide (A3). 3. Saturated Lactones with a |3-Hydroxyi Group (A5) In the recently discovered A5 lactones-those bearing a hydroxyl group on the lactone moiety~the relative configuration within the lactone has been reported by Cortes et alJO In itrabin (7) and jetein (33), the protons on the lactone were found to exhibit ^H NMR nOe's of 2% between H(2) and H(33) and 1% between H(33) and H(34), suggesting an all-cis stnjcture (Figure 18). Coupling constants of -5.5 Hz between H(2) and H(33) and -7 Hz between H(33) and H(34) were measured for jetein 33;36 these data are consistent with the reported configuration.

Figure 18. Examples of p-Hydroxybutanolide-containing (A5) Acetogenins.

276 III. OTHER

FUNCTIONALITY

Although all Annonaceous acetogenlns possess some variant of the lactone subunit, and most have THF rings, other functional groups are often present along the carbon chain (or in place of the THF ring structure in presumed biological precursors). One of these substructures, the carbonyl moiety (C2), adds no stereochemical complexity to the natural product; the rest, however, have stereochemical Issues that must be addressed in determining the complete three-dimensional structures of the natural products. Since most new research has focused on other parts of the acetogenlns, this is an area that is still being explored. However, since these functional groups are not unique to this class of natural products, some classical methods do exist for determining their configuration. In some acetogenlns, a lone hydroxyl group (C1) has been found along the carbon chain. In one such compound, the absolute configuration of this carbinol center has been successfully determined. McLaughlin et. al.^^ prepared the tris-MTPA esters of bullacin (34) and concluded, from the data summarized in Table 9, that C(6) possesses an S configuration. Note that even protons relatively remote from C(6) show useful A5's.

Table 9.

Mosher Ester A5H Data for the Tris-MTPA Derivative of Bullacin (34). 5

H(35)

(S'MTPA'34)

5

(R'MTPA'34)

A5H

= 5S - 6R

1.39

1.39

0

H(34)

4.97

4.96

+ 0.01

H(33)

6.95

6.86

H(3)

2.18

+ 0.09 + 0.09

H(4)

2.27 1.54

H(5)

1.67

H(6) H(7)

5.08 1.57

1.55 5.07 1.67

1.43

+ 0.11 + 0.12 -0 -0.10

277 Some acetogenins, such as rollinicin (35), have vicinal did moieties (C4) in their structure. Sneden et. al.38 employed an interesting method for determining the relative configuration of the diol in rollinicin. By a method previously reported by Murata.^Q the tris-trimethylsilyl ether of the natural product was prepared and analyzed by ci mass spectrometry. According to Murata, if the [MH+ - MeaSiOH - Me2Si=CH2]+ ion (MH - 90 - 72) is in much greater abundance than the [MH+ - MeaSiOH - Me3SiOH]+ ion (MH - 90 - 90), then the relative configuration of the diol is erythro; if the opposite is observed, it is three. With rollinicin, the MH - 90 - 72 ion was found to be twice as abundant as the MH - 90 - 90 ion, suggesting an erythro relationship for the diol In 35.

Another known subunit of the acetogenins is an isolated 1,2-disubstituted alkene moiety (C6). The geometry of the double bond can be determined by inspection of coupling constants In "^H NMR spectroscopy. For example, the vinylic protons, H(9) and H(10), in giganenin (36) were shown to be coupled to each other with J = 10.9 Hz, diagnostic for the Z-olefin geometry.-^o Alternatively, the geometry can be deduced from the •'^C NMR shifts of the allylic carbons. For example, in epomuricenin (37),41 C(18) and C(21) were found to have chemical shifts at 824.0 and 827.0; a trans geometry would have resulted in a downfield shift for these carbons.

37

(Epomuricenin A)

A special case of structure determination is that of muricatacin (38). This unique molecule was isolated from Annona muricata seeds,^ along with more conventional acetogenins, and is considered to be a metabolic product of the plant. By comparing sign and magnitude of optical rotation of 38 with the known, enantiomerically pure analog, (4S,5S)-5-hydroxypentadecan-4-olide (39), an intermediate in the synthesis of disparlure, it was concluded that 38 exists as a mixture of the (4R,5R) and (4S,5S) enantiomers, with the (4f?,5f?) slightly in excess (Figure 19). This hypothesis was later supported by syntheses of both enantiomers of muricatacln.6.42-44

278

38

(MurJcatacin)

[alo^ - +29.2° Mixture of (R,R) and (S,S) isomers

(S.S)

Figure 19. Comparison of Rotation Data for Muricatacin (38) and Analog (39).

IV. VALIDATION OF STRUCTURE DETERMINATION METHODS In several cases, evidence for the validity of the methods described in this chapter has come by chemical conversion of one molecule into another, known product, thus demonstrating that they possess the same relative (and in some cases, absolute) configuration. The first example of such a conversion was the treatment of bullatacin (5) to produce the rearranged product, bullatacinone (6) (Figure 20). The product so obtained proved to be indistinguishable from the natural product.^ These results confirmed the finding that bullatacin and bullatacinone have the same relative configuration of the THF subunit, thus providing support for the method used to make those independent assignments. A similar result was obtained in the conversion of 25desoxy-4-hydroxyneorollinicin (40) to rollinone (32).45

Figure 20. Base-catalyzed Conversion of 4-Hydroxy Butenolide Acetogenlns (A2) Into Rearranged Butanolides (A3).

279 In another experiment, annonacin (3) was oxidatively cleaved with mCPBA to muricatacin (38) (Figure 21), the absolute configuration of which had been established by optical rotatlon.5 This conversion suggested the /? configuration at C(20) of annonacin. This fact was later borne out by the Mosher ester method described earlier in this chapter, lending support to the validity of the method.

^^'.. p H

38 (Muricatacin)

HO

Figure 21. Oxidative Cleavage of Annonacin (3) to Muricatacin (38).

Further proof has come through the recent total synthesis of several Annonaceous acetogenins. The first of these to be constnjcted was (+)-(15,16,19,20,23,24)-/)exep^ uvaricin (19), an unnatural diastereomer of uvaricin (1) reported from our laboratories in 1991.27 Knowing the relative, but not the absolute, configuration among C(15)-C(24) of

HO^

]15

0^ 1

41

(en^Bullatacin)

24

(Reticulatacin)

jP l24

M7

r^'o HO

O

38 (Muricatacin)

•H

Figure 22. Acetogenins for Which Total Syntheses Have Been Described.

280 uvaricin (1), we arbitrarily prepared one of the two possible enantiomers of the THF backbone. This was eventually coupled with an A1 fragment having 36-S configuration, as known for the natural product. The resulting acetogenin had ""H and '•^c NMR data identical with the natural product, but a slightly different optical rotation ([a]D = +11.3° for the natural product and +9.5° for the synthetic product). Definitive proof of the difference between these two molecules came with the Mosher ester derivatization of C(15) in both compounds. The (f?)-MTPA derivative of 1 had different i H and ^^F NMR spectra from the (/?)-MTPA derivative of 19, but Identical spectra to the (S)-MTPA derivative of 19. Since the absolute configuration of 19 was known based on the method of its synthesis, this provided conclusive proof of the relative and absolute configuration of uvaricin (1). Further efforts by several groups have resulted in total syntheses of muricatacin 38,6, 42-44 (-).bullatacin (41) (the enantiomer of the natural product),^^ solamin 42,^^7 and reticulatacin 24,^*7 (Figure 22). All of the above have been synthesized by methods that establish unambiguously their absolute configuration. All of the stereochemical information obtained so far by total synthesis is in complete agreement with the conclusions reached earlier by the methods described in this chapter. V.

CONCLUSION Although tremendous progress has been made in this area, several stereochemical

issues remain unaddressed in the Annonaceous acetogenins. The most obvious of these is the fact that there is still no general way to determine the configuration of C(36) in the A1 subunit, that is, the a,p-unsaturated lactone having no hydroxyl groups in Its vicinity. The absolute configuration at this center has been determined for uvaricin by degradation, but as mentioned earlier, this approach may not be practical in all cases. However, the absence of other stereogenic centers on the A1 subunit and the remoteness of this substituent from other functionality presents no small challenge to development of a more general method. In addition, although stereochemical information for portions of the B3 unit (nonadjacent bis-THF) is available, the complete relative configuration of the subunit as a whole remains unsolved {vide supra). Future work will undoubtedly provide more information for molecules of this structure. Despite these limitations, however, a wealth of information now exists on the stereochemical features of the Annonaceous acetogenins, and the complete relative and absolute configuration has been solved for several members of this class. The tremendous value and potential of NMR-based strategies and arguments is clear. Chemical shift trends rather than coupling constant analysis have proven much more powerful for this class of non-rigid molecules. It Is our hope that this collection of structure elucidation methods will prove valuable in the future study of this important family of natural products.

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