J. Am. Chem. SOC.1992, 114, 10203-10213
10203
Determination of Absolute Configuration of Stereogenic Carbinol Centers in Annonaceous Acetogenins by 'H- and 19F-NMR Analysis of Mosher Ester Derivatives Matthew J. Rieser,? Yu-hua Hui,+ J. Kent Rupprecht,? John F. Kozlowski,t Karl V. Wood,$Jerry L. McLaughlin,*,t Paul R. Hanson,g Zhiping Zhuang,§ and Thomas R. Hoye*,% Contributionfrom the Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, and Department of Chemistry, School of Science, Purdue University, West Lafayette, Indiana 47907, and Department of Chemistry, University of Minnesota, Minneapolis. Minnesota 55455. Received June 22, 1992
Abstract: The absolute configuration of the stereogenic carbinol centers in nine annonaceous acetogenins has been determined by careful IH-and "F-NMR analysis of (9-and (R)-Mosher ester [methoxy(trifluoromethyl)phenylacetate or MTPA] derivatives. These acetogenins include five adjacent bis-tetrahydrofuran acetogenins [uvaricin (3), bullatacin (4), bullatacinone (9,asimicin (6),and rolliniastatin 1 (7)] and four mono-tetrahydrofuran acetogenins [reticulatacin (8), isoannonacin-10-one (9), annonacin-10-one (lo), and annonacin (ll)]. Importantly, the configuration at the C(4) carbinol center in all of the biologically
most significant acetogenins examined here is R. The validity of the refined Mosher methodologyBcfor assigning absolute carbinol stereochemistry in several specific substructural regions of these acetogenins was established by analysis of the MTPA esters of appropriate synthetic model compounds. These possessed unambiguous absolute (as well as relative) stereochemistry. It was demonstrated that the analysis of proton and fluorine NMR data from atoms flanking carbinol centers is always a valid strategy for assigning absolute configuration at any of the carbinol centers, given the availability of suitable model compounds of known absolute configuration. The interpretation of the fluorine NMR data, however, must be performed with caution. Although there are clearly identifiabletrends among the fluorine data, whether they are indicativeof R or S absolute configuration is dependent upon quite subtle local structural features.
Introduction Annonaceous acetogenins are a rapidly growing class of new naturally occurring polyketide-derived fatty acid derivatives isolated from a number of plants in the Annonaceae. A reviewla has been published describing the sources, isolation, chemistry, biogenesis, and biological activitiestbof these new compounds. Because of their multiple stereogenic centers and the likely importance of stereochemistry for their bioactivities, the determination of all the stereochemical issues in this type of compound is an important concern. The waxy, amorphous, or microcrystalline nature of these compounds has, thus far, usually rendered them unsuitable for direct X-ray crystallographic studies.2 Methodology has been developed previously to determine the relative stereochemical relationships among the stereogenic carbon atoms around the THF and a technique has been published previously for determining the relative stereochemistry of a stereogenic carbinol center adjacent to a tetrahydrofuran ring.2b The absolute configuration a t C(36) of uvaricin and desacetyluvaricin has been determined by ozonolysis and subsequent chromatographic comparison with the appropriate derivatives of ( R ) - and (S)-lactic acid.5 The absolute configuration at C(4) for bullatacinone and bullatalicinone has been suggested by comparisons of the CD spectra of these compounds with those of the model compound, r~brenolide.~.'However, there is ambiguity associated with the last of these methods.la Among the reported adjacent bis-tetrahydrofuran acetogenins, at least ten structures (uvaricin, asimicin, bullatacin, rolliniastatin 1, 4hydroxy-25-desoxyneorollinicin,annonin VI,annonareticin, 14hydroxy-25-desoxyrollinicin (revised), rollinicin 2, and laherradurine)Ia have the same skeleton of contiguous carbon atoms. Determination of the relative stereochemistries has differentiated some of the C(15)-C(24) diastereomenc relationships in this series, such as those found in uvaricin, asimicin, bullatacin, rolliniastatin 1, and 4-hydroxy-25-desoxyneorollinicin.In this paper we describe Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University. Department of Chemistry, School of Science, Purdue University. Department of Chemistry, University of Minnesota.
the determination of the absolute configurations of stereogenic carbinol centers in several annonaceous acetogenins using Mosher ester methodology. The Mosher ester technique is an empirically derived method to determine the absolute configurationsa* (and enantiomeric excess)se of stereogenic centers bearing hydroxyl (or amino) groups. The chiral alcohol (or amine) is first coupled with each of the individual enantiomers of the Mosher acid chloride. The optically pure Mosher acid of R configuration [(R)-MTPA] is converted to the acid chloride of S configuration [(S)-MTPA-Cl] and then on to the MTPA ester of R config~ration.~The lH(1) (a) Rupprecht, J. K.; Hui, Y.-H.; McLaughlin, J. L. J . Nut. Prod. 1990,53,237. (b) It has been determined recently that acetogenins such as
asimicin act as inhibitors (IC5,, in the subnanomolar range) of complex I in the mitochondrial electron transport systems: Lewis, M. A,; Arnason, J. T.; Philogene, B. J. R.; Rupprecht, J. K.; McLaughlin, J. L. Pestic. Biochem. Physiol., in press. (2) Exceptions for which single-crystal X-ray data have been successfully obtained are (a) the lS-O(p-bromophenyl)urethane derivative of rolliniastatin 1 (Pettit, G. R.; Cragg, G. M.; Polonsky, J.; Herald, D. L.; Goswami, A,; Smith, C. R.; Meretti, C.; Schmidt, J. M.; Wiesleder, D. Can. J . Chem. 1987, 65, 1433) and (b) the dihydro and lactone-hydrolyzed carboxylate salt of annonin I Born, L.; Lieb, F.; Lorentzen, J. P.; Moeschler, H.; Nonfon, M.; Sollner, R.; Wendisch, D. Plunta Med. 1990, 56, 312). (3) Hoye, T. R.; Suhadolnik, J. C. J . A m . Chem. SOC.1987, 109, 4402. (4) Hoye, T. R.; Zhuang, Z. J . Org. Chem. 1988, 53, 5578. (5) (a) Jolad, S.D.; Hoffmann, J. J.; Schram, K. H.; Cole, J. R.; Tempesta, M. S.;Kriek, G. R.; Bates, R. B. J . Org. Chem. 1982, 47, 3151. (b) Jolad, S. D.; Hoffmann, J. J.; Cole, J. R.; Barry, C. E., 111; Bates, R. B.; Linz, G. S.;Konig, W. A. J . Nut. Prod. 1985, 48, 644. (6) Hui, Y.-H.; Rupprecht, J . K.; Liu, Y.-M.; Anderson, J. E.; Smith, D. L.; Chang, C.-J.; McLaughlin, J. L. J . Not. Prod. 1989, 52, 463. (7) Hui, Y. H.; Rupprecht, J. K.; Anderson, J. E.; Wood, K. V.; McLaughlin, J. L. Phytother. Res. 1991, 5 , 124. (8) (a) Dale, J . A.; Mosher, H. S. J . A m . Chem. Sac. 1973, 95, 512. (b) Sullivan, G. R.; Dale, J. A.; Mosher, H. S.J . Org. Chem. 1973, 38, 2143. (c) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J . Am. Chem. Sac. 1991, 113, 4092. (d) For reasons other than determination of absolute stereochemistry, the recommendation has been made to routinely record MTPA data on all newly isolated acetogenins: Fujimoto, Y.; Murasaki, C.; Kakinuma, K.; Eguchi, T.; lkekawa, N.; Furuya, M.; Hirayama, K.; Ikekawa, T.; Sahai, M.; Gupta, Y. K.; Ray, A. B. Tetruhedron Lett. 1990, 31, 535. (e) Dale, J. A.; Dull, D. L.; Mosher, H. S.J . Org. Chem. 1969, 34, 2543.
0002-7863/92/1514-lO203$03.00/00 1992 American Chemical Society
10204 J. Am. Chem. Soc., Vol. 114, No. 26, 1992
Rieser et ai.
Table I. 'H-NMR Chemical Shift Data' for H(14)-H(25) from the (S)and (R)-Per-MTPA Mosher Ester Derivatives of the (Synthetic) Model Bis-Tetrahydrofuran 12, (Synthetic) hexepi-Uvaricin (13), and the (Natural) Acetogenins Containing th/t/th/t/ ... Relative Stereochemistry among C( 15)-C(24) (3-6) and the Mono-Mosher Ester Derivatives of 5 and 12
.. ..
I
I
mono-MTPA Derivative (carbinol)
I 1
Model bis-THF (12) 15-mono-MTPAester
1 83
S R
Bullatacinone (5) 15-MTPA ester
R
Bullatacinone(5) 24-MTF'Aester
R
11,591 'Os
1
S d
S
4.05 4.131 neg
:::;:]
1
-zero
d
1
3.60/3.8 1-3.87 3.81-3.87 1.37 3.80D.89-3.921neg 3.893921neg/ 1.371 zero d
I
d
d
4.03
d
1
I R
S
R
S
"All chemical shifts > 6 2.0 are determined directly from the ID 'H-NMR spectrum whereas, because of the large degree of overlap among the resonances, all shifts < 6 2.0 are carefully estimated from the center of the relevant COSY off-diagonal peaks. bNot applicable since the C(24) hydroxyl is acetylated in 13 and 3. CEffectsfrom both ends are in opposition. d ' H / l H COSY data were not acquired.
1
2
-
Figure 1.
NMR and I9F-NMR spectra of the Mosher ester derivatives are then obtained. The absolute configuration of the carbinol (or amine) center can then be deduced by comparison of the chemical shifts in the IH-NMR or 19F-NMR spectra of the (R)-MTPA and (S)-MTPA esters. The recent refinement of the Mosher method developed by Kakisawa and KashmansCpoints out the advantages of analysis based upon the more reliable proton NMR chemical shifts as well as ambiguity inherent in the use of fluorine chemical shift data for the assignment of configuration. The Mosher argument as(9) A referee has commented on the possible confusion that arises over a nomenclature issue first clearly noted by Mosher (ref 8a, footnote 46). Namely, Cahn-lngold-Prelog priority interchange accompanies each of the chemical conversions of the MTPA acid to MTPA-CI and of MTPA-CI to the MTPA ester. That is, the S acid (i) gives rise to the S ester (iii) but by way of the R acid chloride (ii), and vice versa. This is a particularly crucial detail now that both enantiomers of MTPA-CI (as well as the MTPA acid) are commercially available (e&, Aldrich Catalog, 1992-1993). MeqCF, MeOCF, ROH MeqCF, PhXC,"
i
-
S(-)-MTPA-acid [a10 -70' (c = 1.6, MeOH)
-
P
h
L
-
Ph&R ,
ii
iii
R(-).MTPA-CI [a10 -131 (c I 6.1, CHCI,)
SMTPA-ester
-
sumes that the most stable conformation of the (S)-MTPA ester is as shown in 1 (Figure 1). Protons in the L3 portion of this molecule are, therefore, more highly shielded (and appear further upfield), and those in the L2moiety are less highly shielded (and appear further downfield). The reverse is true for the (R)-MTPA ester 2 (Figure 1). The difference in chemical shift of any set of like protons in the diastereomeric (S)and (R)-MTPA esters (AbH = bS -)6, will therefore be positive in value when associated with L2 and negative in value when associated with L3. This method gives greater reliability, in part, because modern NMR techniques often permit the identification of a number of like sets of protons within L*,thereby reinforcing the assignment made by a comparison of the chemical shifts of only a single set.8c In contrast the fluorine shift data necessarily allow only a single point comparison. Moreover, it requires that one know confidently which of the diastereomers 1 and 2 has a greater population of the most stable conformation having the CF3 group in the deshielding plane of the carbonyl (as shown in Figure l). The relative energies of the various conformers generated upon rotation about the carbonyl carbon to benzylic carbon bond, in turn, is dependent upon sometimes subtle differences in through-space steric (and electronic) effects between Lnand Ph or MeO. We have used this refined Mosher ester strategysCto examine the absolute stereochemistries of certain stereogenic centers of several annonaceous acetogenins [uvaricin (3), bullatacin (4), bullatacinone (5), asimicin (6), rolliniastatin 1 (7), reticulatacin (8), isoannonacinon- 10-one (9), annonacin-l O-one (IO), and annonacin (ll)]and describe the results here. Results and Discussion MTPA Proton Data for Carbinol Centers Flanked by THF Rings: Validation of Synthetic Model Compounds and Application to Uvaricin. Uvaricin (3), bullatacin (4), bullatacinone (5), and asimicin (6)[and rolliniastatin 1 (7)] (Figure 2) belong to the subgroup of adjacent, or 2,5-linked, bis-tetrahydrofuran acetogenins. Each of 3-6 is now (vide infra for 4 and 5) known to
Absolute Configuration of Acetogenins
I'""'"[
J . Am. Chem. SOC., Vol. 114, No. 26, 1992 10205
Skeletal Numbering
e thrm
Asimian 6
%+ trans
e erythro
O+
threo
erythro
Me
HO
HO"'
Rolliniastatin 1
7
cis
HO"'
Me
HO
HO
Me
Me
AcO
Figure 2.
have relative stereochemical relationships of threo between C( 15) and C(16), trans between (or across) C(16) and C(19), threo between C(19) and C(20), and trans between (or across) C(20) and C(23). These relationships are denoted by th/t/th/t/er for 3-5 and th/t/th/t/th for 6. Preliminary verification of the Mosher methodology to this substructural unit was obtained by analysis of the two synthetically prepared bis-tetrahydrofurans 12'Oa and 13Iob(15,16,19,20,23,24-hexepi-uvaricin) (Figure 2). Because of the means of their preparation, these two compounds possess the unambiguous relative and absolute stereochemistry shown. Relevant proton NMR data for the bis-(R)- and bis-(a-Mosher esters of the C2-symmetric bis-THF diol 12 having er/t/th/t/er relative stereochemistry are summarized in entry 1 of Table I. Note that the carbon skeleton numbering for 12 (as well as for other model compounds described later) follows that of uvaricin and the other bis-THF acetogenins. The synthetic derivatives S-MTPA-12 and R-MTPA-12 are known to have the R configuration at both C(15) and C(24). Therefore, the methylene protons H( 14) reside in L2 (cf. Figure l), and the methine protons H(16) and H(19) lie within L3.As shown in Figure 3, the sign of ASH [=(as - bR)]is therefore positive for H(14) [and H(25)], negative for H(16) [and H(23)], and negative for H(19) [and H(20)]. In other words, H(16) and H(19) are relatively more shielded, and H( 14) is relatively less shielded, in S-MTPA-12 because of their various proximities to the phenyl ring. All of the proton N M R data reported here were collected at 500 MHz. A comparison of a few random spectra at both 500 and 300 MHz suggested that the great majority of information can be extrapolated at both field strengths, even when the AS (10) (a) Preparedanalogously to the compound numbered l l c in Hoye and Suhadolnik (Hoye, T.R.; Suhadolnik, J. C. Tetruhedron 1986,42, 2855); Le., the bis primary tosylate related to the compound numbered 5 in ref 10b was coupled with lithium di-n-nonylcuprate. (b) Hoye, T.R.; Hanson, P. R.; Kovelesky, A. C.; Ocain, T.D.; Zhuang, Z. J . Am. Chem. Sor. 1991, 114, 9369.
. .
.___..._. _._ +0'04 ----- _-_. S 1.62-1.54
S-MTPA-12
R -MTPA-12
14
EH,H&Hz(CH,),CH3
8 1 66.1 58
Figure 3.
14
EH,HBCH~(CH~),CH~ I
El A 6H(14)
values are as small as -0.02 ppm. One potential limitation to 300-MHz data is that the additional field dispersion is very useful in permitting confident assignment of small chemical shift differences of protons residing in the aliphatic envelope [Le., H(5), H(14), H(17-18), H(21-22), and H(25)]. These were identified by IH-IH COSY techniques. For synthetic hexepi-uvaricin (13)Iob (entry 2, Table I), which has the unambiguous relative and absolute stereochemistry shown in Figure 2, C( 15) is of S configuration. As expected, then, ASH for H(14) is negative and ASH for H(19)/H(2) is positive. Unexpectedly, ASH for H(16) is -0 (vide infra). [H-NMR data arising from the C(14)-C(25) region of the MTPA derivatives of natural uvaricin (3) (entry 3, Table I) clearly indicate the mirror image relationship between the C( 14)-c(25) portions of uvaricin and hexepi-uvaricin (13). In fact, it was only upon comparison of these MTPA data from 3 and 13 that their stereochemical differences were revealed.lob That fact, in turn, permitted the entire stereo structure of uvaricin to be assigned as 3 for the first time.
Rieser et al. TAble 11. I9F-NMR Chemical Shift Data for CF3(15) and CF3(24) from the (S)-and (R)-Per-MTPA Mosher Ester Derivatives of the (Synthetic) Model Bis-Tetrahydrofuran 12, (Synthetic) hexepi-Uvaricin (IJ), and the (Natural) Acetogenins Containing Relative Stereochemistry among C(15)-C(24) (3-6) th/t/th/t/ and the C( 15)- and C(24)-Mono-Mosher Ester Derivatives of 5
...
carbinol
Derivative
R -MTPA-16th
R-MTPA-14-er
Figure 4.
That A6H for H(16) in both MTPA-3 and MTPA-13 is 0 was surprising. It was therefore necessary to identify a set of compounds of known stereochemistry which would be suitable to model these circumstances. Thus, we prepared the (R)-Mosher esters R-MTPA-14-th and R-MTPA-14-er from a racemic mixture of each of the two (threo and erythro) diastereomers of the corresponding mono-tetrahydrofuran carbinols (Figure 4).Ilaqb The A6H for H(16) in the C(15)/C(16)-threo diastereomers RMTPA-14-th is also approximately 0 (overlapping resonances in the spectrum of the mixture, with A6H < 0.02 ppm). Although the reason(s) for a -0 value of A6H for H( 16) in the threo isomers is not clear, it is reassuring that this outcome is reproduced in the model compounds. Incidentally, note that there is good correlation of the remaining A6H values shown in Figure 4 with the relevant H(14) and H(16) data in entries 1-3 of Table I. This point is further supported by data obtained from the mono-MTPA ester derivatives of 12 and bullatacinone (5) (entries 7-9). Once again, the A6H for H( 16) having a C( 15)/C( 16)-erythro relationship and H(23) having a C(23)/C(24)-erythro relationship is 0.08 (entries 7 and 9), whereas the A6H for H ( 16) having a C( 15)/C( 16)-threo relationship is -0 (entry 8). MTPA Fluorine Data for All Carbinol Centers Flanked by THF Rings. Fluorine N M R analysis has frequently been used for making assignments of the configuration of MTPA estersEb However, Kakisawa and Kashman have cautioned that conclusions based on I9F-NMR analysis are often in error and must be scrutinized with careaECThe fundamental arguments are again based on the assumption of the preferred conformations 1 and 2 (Figure 1). The trifluoromethyl group prefers the illustrated rotamer, in which it is eclipsed with the carbonyl group and is, therefore, within its deshielding cone. The relative extent of this preference for conformers 1 and 2 is assumed to be largely dependent upon the magnitude of the steric interaction between the large phenyl group and the carbinol substituents L2 and L3. For example, if L3 in 1 and 2 is larger than L2, the destabilizing L3/phenyl interaction in conformer 1 of the ( 8 - M T P A ester will result in the trifluoromethyl group spending less time in the carbonyl deshielding plane (relative to conformer 2 for the for the CF3 (R)-MTPA ester). Consequently, A6F [=(6, - a,)] resonance in the I9F-NMR spectra will be negative. Alternatively, if L3 in 1 and 2 is smaller than L2, A6F will be positive. The 19F-NMR data at C(15) and C(24) for the MTPA ester derivatives of 12, 13, and 3-6 are summarized in Table 11. In synthetic hexepi-uvaricin (13) the configuration at C( 15) is S . Thus, L3contains the smaller, unbranched C( 14)-C( 1) subunit, and L2 comprises the larger, a-branched bis-THF moiety commencing with C( 16). As expected from the arguments presented in the last paragraph, A6F is positive for CF3(15) in the MTPA esters of 13 (entry 2, Table 11). Similarly, the MTPA esters of synthetic, C2-symmetric model compound 12 have the R configuration at C(15) and C(24). Accordingly, the A6F values are negative (entry 1, Table 11). Thus, I9F-based assignments of configuration of those carbinol centers directly flanking a T H F ring in these systems are valid. ( I 1) (a) Unpublished results of P. R. Hanson. (b) Details of the synthesis of the carbinol precursors to 14-th and 14er will be reported elsewhere. (c) Unpublished results of Z. Zhuang. (d) Details of the synthesis of 15 and its related stereoisomers will be reported elsewhere.
1 1 I'1 1 I
DenvativesofS
Bullatacinone (5) 15-MTF'Aester
I
I I ::::}I S
R
neg
I
I
Bullatacinone(5) 24-MTPA ester
'Not applicable since the C(24) hydroxyl is acetylated in 13 and 3.
The A6F for uvaricin (3) MTPA esters is negative, reinforcing the C(15) R assignment (entry 3, Table 11). Likewise, the negative A6F values in bullatacin (4), bullatacinone (5), and asimicin (6) MTPA esters suggest an associated R configuration, and the postive A6F values an S configuration, a t the pertinent carbinol centers (entries 4-8, Table 11). MTPA Proton Data for Carbinol Centers Flanking THF Rings in Bullatacin (4), Bullatacinone (5), and Asimicin (6). Bullatacinone (5) and bullatacin (4) contain nonidentical vicinal stereochemical relationships between C( 15) and C( 16) and between C(23) and C(24).6 Which of these is erythro and which is threo (i.e., the "endedness", or distinction between er/t/th/t/th and th/t/th/t/er) was previously ambiguous for these unsymmetrical C(15), C(24)-diols. In the course of generating and interpreting N M R data for the bis- and tris-MTPA esters of 5 and 4, respectively, it became useful to prepare various bis- and monoMTPA esters, respectively. After separation it was possible to determine the p i t i o n of esterification by analysis of mass spectral fragmentation patterns, which are summarized in Figure 5a-c for C( 15),C(24)-bis- and C( 15)- and C(24)-mono-MTPA esters of bullatacinone and in Figure 6a-c for C(4),C( 15),C(24)-trisand C(4),C( 15)- and C(4),C(24)-bis-MTPA esters of bullatacin, respectively. These clearly establish the positional nature of these partial MTPA derivatives. Analysis of I3C- and 'H-NMR data from some of these derivatives unambiguously allowed the assignment of the relative configurations between C( 15) and C( 16) as threo [unesterified carbinol carbon and proton resonances for, e.g., the ester shown in Figure 5b: 6 71.45, C(24), and 3.8-4.0, H(24)]11as12 and between C(23) and C(24) as erythro [unesterified carbinol carbon and proton resonances for, e.g., the ester shown in Figure 5c: 6 74.03, C(15), and 3.37, H(15)].'1a-12Thus, the C( 15)
