Nodulisporic acid A, a novel and potent insecticide from a nodulisporium Sp. isolation, structure determination, and chemical transformations

Article abstract of DOI:10.1021/ja971664k

The potent insecticidal agent nodulisporic acid A (1a), representative of a new class of indole terpenes, was isolated from fermentations of a Nodulisporium sp. Nodulisporic acid A was active against the larvae of the blowfly and mosquito at sub-part-per-

Full text of DOI:10.1021/ja971664k

J. Am. Chem. Soc. 1997, 119, 8809-8816
8809
Nodulisporic Acid A, a Novel and Potent Insecticide from a
Nodulisporium Sp. Isolation, Structure Determination, and
Chemical Transformations
John G. Ondeyka,* Gregory L. Helms,* Otto D. Hensens,* Michael A. Goetz,
Deborah L. Zink, Athanasios Tsipouras, Wesley L. Shoop, Lyndia Slayton,
Anne W. Dombrowski, Jon D. Polishook, Dan A. Ostlind, Nancy N. Tsou,
Richard G. Ball, and Sheo B. Singh*
Contribution from Merck Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065
ReceiVed May 21, 1997X
Abstract: The potent insecticidal agent nodulisporic acid A (1a), representative of a new class of indole terpenes,
was isolated from fermentations of a Nodulisporium sp. Nodulisporic acid A was active against the larvae of the
blowfly and mosquito at sub-part-per-million levels. The structure followed mainly from a consideration of
spectroscopic evidence, including Dunkel’s computerized 2D INADEQUATE analysis. The relative stereochemistry
of the eastern and western hemispheres of 1a and its methyl ester, 1b, were independently determined on the basis
of ROESY, NOESY, NOEDS, and Jvic evidence. By employing the same methods, the complete relative
stereochemistry was determined by analysis of suitable transformation products, using the reduced â-ketodihydropyrrole
ring as a stereochemical bridge between the two zones. These results were confirmed by X-ray analysis of the
7-p-bromobenzoate methyl ester derivative, 1c. The absolute stereochemistry was established by application of the
advanced Mosher method to methyl ester 1b. Of biogenetic interest is the presence of a unique isoprenylated indole
moiety not previously found in other indole mycotoxins.
The development of resistance to existing insecticides and
the desire to have compounds with less mammalian and
environmental toxicity continues to spur the search for novel
insecticides. Screening culture extracts using the larvae of the
mosquito, Aedes aegypti, has been well documented as a viable
approach to discovering new insecticidal activities.1a A unique
macrocyclic lactone, spinosyns, now under development as a
crop protection agent, was detected using the mosquito larvae
assay and was isolated from Saccharopolyspora spinosa.1c
Another very useful assay involves the larvae of the blowfly,
Lucilia seracata. Either of the assays can be used to measure
insecticidal activities of agents such as avermectins or
milbemycins.1b,d In our ongoing screening program for biologi-
cally active natural products, we have isolated a novel indole
terpene designated nodulisporic acid A (1a) from a Nodulispo-
rium sp. as a potent insecticide. We describe herein the isolation
and structure elucidation of 1a, including the relative and
absolute stereochemistry, solution conformation, and biological
activity. A number of chemical transformations are also
described.
chromatographed over silica gel, followed by a gel filtration
step (Sephadex LH20). Final purification was achieved by
preparative reverse-phase HPLC, providing nodulisporic acid
as a yellow powder in 2 mg/L yield.
Structure Determination. High-resolution FAB-MS of
nodulisporic acid A indicated the molecular formula C43H53-
NO6 (found m/z 680.3991, calcd m/z 680.3951 [M + H]), which
was supported by the carbon and carbon-bound proton counts
from 13C NMR and DEPT spectra, respectively, assuming three
exchangeable protons. The molecule contains one ketone
carbonyl, one carboxylic acid group, and eight double bonds
(only one of which is isolated), suggesting eight rings to satisfy
the 18 double bond equivalents. Three partial structures, A-C
(Figure 1), were proposed on the basis of the COSY, TOCSY,
HMQC, and HMBC data, which left seven carbons unaccounted
for, six of which were non-protonated (see Table 1).
Three exchangeable groups (OH at C7 and C24 and C5′′
COOH) were detected by application of the technique involving
deuterium isotope-induced 13C shifts (in CD2Cl2 spiked with
D2O), which characteristically affect2 those carbons R and â to
NH/OH groups in alcohols and amines. Additionally, nod-
ulisporic acid A (1a), on reaction with diazomethane at -78
Results and Discussion
Isolation. The Nodulisporium sp. (MF 5954, ATCC 74245)
is an endophytic fungus isolated from a woody plant. Nod-
ulisporic acid A could be purified from methyl ethyl ketone
extracts of the fungus grown either on a solid substrate or in a
liquid medium. The extract was dried and partitioned between
hexane and MeOH (1:1). The MeOH solubles were first
X Abstract published in AdVance ACS Abstracts, September 15, 1997.
(1) (a) Henrick, C. A.; Staal, G. B.; Siddall, J. B. J. Agric. Food Chem.
1973, 213, 354. (b) Putter, I.; MacConnell, J. G.; Prizer, F. A.; Haidri, A.
A.; Ristich, S. C.; Dybas, R. A. Experientia 1981, 37, 963. (c) Kirst, H.
A.; Michel, K. H.; Mynderse, J. S.; Chio, E. W.; Yao, R. C.; Nakasukasa,
W. M.; Boeck, L. D.; Occlowitz, J. L.; Paschal, J. W.; Deeter, J. B.;
Thompson, G. D. ACS Symp. Ser. 1992, 504, 214-225. (d) Shaw, R. D.;
Blackman, G. G. Aus. Vet. J. 1971, 47, 268.
S0002-7863(97)01664-8 CCC: $14.00 © 1997 American Chemical Society

8810 J. Am. Chem. Soc., Vol. 119, No. 38, 1997
Ondeyka et al.
fragments A-C (Figure 1). By default, the nitrogen must,
therefore, be placed adjacent to the allylic C2′ carbon in
fragment B to account for the H2′ chemical shift (δ 5.11) and
the corresponding 13C resonance (δ 76.4). A significant number
of plausible structures were generated, of which 1a appeared
the most likely on the basis of the two- and three-bond HMBC
correlations (optimized for 7 Hz) and 13C chemical shift
assignments (Table 1). The only carbon not assigned by
analysis of the HMBC data was the signal at 121.8 ppm; this
could be ascribed to the indole â carbon C15 because of its
similar chemical shift to C14 (122.7 ppm). Moreover, the
structure adequately accounts for the low-field position of C27
(162.0 ppm), which is simultaneously R to the indole nitrogen
and â to the carbonyl group. This assignment was further
substantiated by its HMBC correlation to H2′. Of interest is
the exchange-broadening phenomenon involving the 1H and 13
C
resonances of the isopropenyl group at C2′ but no longer present
in the 2′-epimeric compound 2 (see Solution Conformation,
below).
Figure 1. Partial fragments A-C of nodulisporic acid A (1) mainly
from COSY/TOCSY (bonds in boldface) and HMBC/HMQC data (see
Table 1).
Table 1. 1H and 13C NMR Data of Nodulisporic Acid A (1a) in
CD2Cl2
position
δCa
δHb
HMBC C no.c
1
2
154.7
3
4
5
6
7
8
9
10
11
12
13
56.1
39.1
32.3
25.95
76.8
47.8
45.2
24.7
25.7
48.0
27.75
1.93 (m), 1.76 (m)
1.78 (m), 1.81 (m)
3.45 (m)
During the course of NMR experiments in CD3CN, it was
observed that 1a degrades to two compounds, 3a and 3b, as
seen by both NMR and HPLC. After isolation by preparative
HPLC, the structure of the major transformation product, 3a,
was determined by the previous NMR protocol. All proton and
1.65 (m)
1.47 (m), 1.47 (m)
1.55 (m), 1.49 (m)
2.85 (m)
13
R: 2.33 (dd, J ) 10.8, 13.9 Hz) 11, 12
â: 2.76 (dd, J ) 6.5, 13.9 Hz) 2, 3, 12,
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
1′
122.7
121.8
116.7
134.0
135.9
122.0
72.6
7.73, (s)
14, 18, 25, 27
6.07 (d, J ) 3.2 Hz)
17, 20, 23, 31, 32
73.9
58.2
75.3
138.4
113.1
162.0
15.1
19.6
11.24
29.9
31.95
23.45
30.1
2.82 (dd, J ) 3.0, 6.2 Hz)
5.24 (d, J ) 6.2 Hz)
17, 22, 24, 25, 33, 34
18, 22, 25, 26
carbon resonances of 1a were readily accounted for in the
spectra of 3a, except for two of the quaternary sp2 carbons,
which had moved significantly downfield to 174.5 (C2) and
197.8 ppm (C14), indicative of amide and ketone carbonyl
functionalities. 1H NMR comparison of 3a and 3b showed the
products to differ only in the dienoic acid side chain. Whereas
the 1′′E,3′′E configuration is retained in 3a, it is isomerized to
the 1′′E, 3′′Z geometry in 3b. Both products are the result of
oxidation of one of the indole double bonds (C2-C14) in a
manner similar to that previously reported for paxilline3 and
shearinine B4 and recently observed in our laboratory with a
paxilline derivative by allowing it to stand in CD2Cl2 indefinitely
at -20 °C. The mode of cleavage and in-depth structure
0.97 (s)
1.15 (s)
1.07 (s)
1.34 (s)
1.31 (s)
1.13 (s)
1.43 (s)
2, 3, 4, 12
3, 4, 5, 9
7, 8, 9, 1′′
19, 20, 32
19, 20, 31
22, 23, 34
22, 23, 33
198.0
2′
3′
4′
5′
76.4 (br) 5.11 (s)
140.0 (br)
117.5 (br) 5.23 (br s), 4.99 (br s)
18.1 (br) 1.48 (br s)
27, 1′, 3′, 4′
2′, 5′
2′, 3′, 4′
7, 8, 9, 30, 3′′
8, 3′′, 4′′
1′′, 2′′, 4′′, 5′′, 6′′
1′′ 154.6
2′′ 125.9
3′′ 140.8
4′′ 125.1
5′′ 172.6
5.96 (d, J ) 15.2 Hz)
6.42 (dd, J ) 11.2, 15.2 Hz)
7.34 (br d, J ) 11.2 Hz)
(2) (a) Hansen, P. E. In Progress in NMR Spectroscopy; Elmley, J. W.,
Feeney, J., Sutcliffe, L. H., Eds.; Pergamon Press: Oxford, 1983; Vol. 15,
pp 105-234. (b) Hansen, P. E. In Progress in NMR Spectroscopy; Elmley,
J. W., Feeney, J., Sutcliffe, L. H., Eds.; Pergamon Press: Oxford, 1988;
Vol. 20, pp 207-255.
(3) (a) Mantle, P. G.; Burt, S. J.; MacGeorge, K. M.; Bilton, J. N.;
Sheppard, R. N. Xenobiotica 1990, 20, 809. (b) Springer, J. P.; Clardy, J.;
Wells, J. M.; Cole, R. J.; Kirksey, J. W. Tetrahedron Lett. 1975, 2531.
(4) Belofsky, G. N.; Gloer, J. B.; Wicklow, D. T.; Dowd, P. F.
Tetrahedron 1995, 51, 3959.
6′′
12.65
1.96 (d, J ) 1.2 Hz)
3′′, 4′′, 5′′
a 125 MHz. 500 MHz. c Optimized for J ) 7 Hz.
b
°C, formed exclusively a monomethyl ester (1b), which
confirmed the presence of a carboxyl group.
These experiments account for all six oxygens in the three

Studies on Nodulisporic Acid A Insecticide
J. Am. Chem. Soc., Vol. 119, No. 38, 1997 8811
Relative and Absolute Stereochemistry and Solution
Conformation of Nodulisporic Acid A by NMR. Noduli-
sporic acid A (1a) contains two separate stereochemical
hemispheres. The relative stereochemistries of the eastern and
western hemispheres of 1a were independently determined on
3
the basis of ROESY, NOESY, NOEDS, and Jvic evidence.
Nodulisporic acid A (1a) and the derivatives revealed strong
NOESY correlations at 400 MHz but very weak correlations at
500 MHz. This is a result of the frequency dependence of the
NOE intensity in the particular molecular weight range of these
compounds (679-900), which at 500 MHz falls in the NOE
zero-crossing region from positive to negative. As ROEs are
always positive, the largely complementary NOESY and
ROESY data of 1a were collected at 400 and 500 MHz,
respectively.
(a) Relative Stereochemistry of Eastern Hemisphere. The
trans-anti-trans fusion of the three cyclic rings was deter-
mined on the basis of ROESY and NOESY correlations of 1a
and 1b (Figure 4). The correlations between H9 and both H7
and 28-H3 established their 1,3-diaxial relationship. The angular
methyl 29-H3 showed strong ROE/NOE correlations to H12 and
30-H3, also confirming their 1,3-diaxial configuration. The
olefinic side-chain proton H1′′ gave strong correlations to H7ax,
H9ax, and H3′′, thus placing the 7-OH and the side chain at the
respective equatorial positions. The H13â methylene exhibited
NOE correlations to H12, whereas H13R gave correlations to
28-H3.
(b) Relative Stereochemistry of Western Hemisphere. The
relative stereochemistry of the eastern hemisphere cannot be
directly correlated to that of the western hemisphere because
of their separation by a number of sp2 centers. The problem is
all the more challenging as the two vicinal asymmetric (C23/
C24) centers are located in a five-membered ring, where
coupling constants are seldom definitive in determining relative
configurations. The NOE/ROE correlations from the methyl
groups to H23/H24 or Vice Versa could not be relied on for the
H23/H24 stereochemical assignment due to the conformational
flexibity of the dihydropyran ring. The vicinal coupling constant
(J ) 6.2 Hz) between H23 and H24 was not diagnostic, since
the predicted J value for both cis and trans hydrogens is the
same. Therefore, it became important to find a derivative which
not only allows correlation of the stereochemistry at C24 to
the eastern hemisphere but also provides a handle for establish-
ing its orientation with respect to H23. Encouraged by the
observation of NOEs from H16 to both H19 and H13 in the
northern half of the molecule, we reasoned that a hydrogen
introduced at C1′ in the southern half (preferably by nonste-
Figure 2. 13C-13C bonds (in boldface) in 1a directly detected by the
INADEQUATE analysis.
elucidation of 3a provides independent support for the proposed
structure (1a) of nodulisporic acid A. Sensitivity toward light
and oxygen, apparently independent of the nature of the solvent,
accounts for these oxidation reactions.
INADEQUATE Analysis. To complete the spectroscopic
structure determination of 1a, several INADEQUATE experi-
ments were conducted to directly detect 13C-13C connectivities
in the molecule to confirm its carbon skeleton. Both natural
abundance and 13C-enriched material were used in these
experiments, and the spectral data were subjected to the
computerized CCBond analysis technique proposed by Dunkel
and colleagues.5 The procedure detects the absence or presence
of bonds between a pair of 13C nuclei whose double-quantum
frequencies can be generated from the 13C frequencies obtained
from a simple 1D 13C NMR experiment. The dramatically
reduced sample requirement of the method is a distinct
advantage under conditions when signals are too weak to be
identified visually.6 In the natural abundance case, 32 out of a
possible 47 connectivities were identified on 26 mg of 1a in
3-5 day experiments optimized for a low (40 Hz) and high
JCC value (60 Hz) using a 3 mm dedicated 13C microprobe.
Several milligrams of doubly labeled 13C compound were
isolated from a fermentation experiment, using doubly labeled
[13C] acetate. Despite a wide range of incorporation efficiencies
(0.1-1.0%) at different sites in the molecule, only two additional
connectivities were found in the INADEQUATE experiment,
one being the C1′-C2′ bond. All bonds detected in this manner
are consistent with the proposed structure. Figure 2 depicts a
composite drawing of all bonds in 1a detected with both the
natural abundance and the 13C-enriched material.
Structure and Relative Stereochemistry of C7 p-Bro-
mobenzoate Derivative 1c by X-ray Crystallography. Two
independent approaches (NMR and X-ray) were simultaneously
taken to deduce the relative stereochemistry of nodulisporic acid
A. The X-ray is described here, and the NMR approach follows.
The methyl ester 1b was reacted with 1.5 equiv of p-
bromobenzoyl chloride in the presence of 4-(dimethylamino)-
pyridine (DMAP) in CH2Cl2 to give 1c in excellent yield. No
epimerization was observed in the absence of CH3CN as a
solvent with the DMAP concentration kept low. Crystallization
of 1c from MeOH-CH2Cl2 at room temperature gave crystals
suitable for X-ray crystallography. The structure and the relative
stereochemistry of nodulisporic acid were confirmed by an X-ray
crystallographic analysis7 of crystals of 1c (Figure 3). Due to
poor crystal quality, however, the absolute configuration could
not be independently established.
(7) Crystal structure details for 1c: C51H60BrNO8, Mr ) 894.958,
monoclinic, space group C2, a ) 38.26(1), b ) 9.709(2), and c ) 12.729-
(4) Å, â ) 99.52(3)°, V ) 4664(4) Å3, Z ) 4, Dx ) 1.275 g cm-3
monochromatized radiation λ(Cu KR) ) 1.541 838 Å, µ )1.61 mm-1
,
,
F(000) ) 1888, T ) 294 K. Data were collected on a Rigaku AFC5
diffractometer to a θ limit of 71.39°. There are 4705 unique reflections out
of 4779 measured, with 2380 observed at the I g 2σ(I) level. The structure
was solved by direct methods (SHELXS: Sheldrick, G. M. Acta Crystallogr.
1990, A46, 467-473) and refined using full-matrix least-squares
(SHELXL: Sheldrick, G. M. J. Appl. Crystallogr., to be published) on F2
using 543 parameters and all data (4705 reflections). All non-hydrogen atoms
were refined with anisotropic thermal displacements, except for the oxygen
of the water molecule, which was left as isotropic. Hydrogen atoms are
included at their calculated positions and were allowed to “ride” during
refinement. Final agreement statistics are R ) 0.070, wR ) 0.180, S )
1.08, (∆/σ)max ) 0.32. The maximum peak height in a final difference
Fourier map is 0.615 e Å-3, and it has no chemical significance. Presumably
as a result of poor crystal quality, the refinement results could not be used
to unambiguously assign the absolute configuration. The authors have
deposited the atomic coordinates for this structure with the Cambridge
Crystallographic Data Centre. The coordinates can be obtained on request
from the Director, Cambridge Crystallographic Data Centre, 12 Union Rd.,
Cambridge CB2 1EZ, UK.
(5) (a) Dunkel, R.; Mayne, C. L.; Curtis, J.; Pugmire, R. J.; Grant, D.
M. J. Magn. Reson. 1990, 90, 290. (b) Dunkel, R.; Mayne, C. L.; Pugmire,
R. J.; Grant, D. M. Anal. Chem. 1992, 64, 3133. (c) Dunkel, R.; Mayne, C.
L.; Foster, M. P.; Ireland, C. M.; Li, D.; Owen, D. L.; Pugmire, R. J.; Grant,
D. M. Anal. Chem. 1992, 64, 3150.
(6) (a) Foster, M. P.; Mayne, C. L.; Dunkel, R.; Pugmire, R. J.; Grant,
D. M.; Kornprobst, J.-M.; Verbist, J.-F.; Biard, J.-F.; Ireland, C. M. J. Am.
Chem. Soc. 1992, 114, 1110-1111. (b) Perera, P.; Andersson, R.; Bohlin,
L.; Andersson, C.; Li, D.; Owen, N. L.; Dunkel, R.; Mayne, C. L.; Pugmire,
R. J.; Grant, D. M.; Cox, P. A. Magn. Reson. Chem. 1993, 31, 472.

8812 J. Am. Chem. Soc., Vol. 119, No. 38, 1997
Ondeyka et al.
Figure 3. Perspective ORTEP drawing of X-ray structure of 1c.
Figure 5. Selected NOESY/NOEDS correlations of western hemi-
sphere of 5a.
Figure 4. Selected ROESY/NOESY correlations in nodulisporic acid
A (1a/1b).
-8°) in the minimized structure (ChemDraw 3D). The 2.8 Hz
coupling constant between H1′ and H2′ of the epimeric
compound 5c is consistent with the measured φ of 113° and,
together with the 3 Å interatomic distance, readily accounts for
the weak NOE between them. A strong syn oxygen effect on
the 5′-CH3 group (downfield shift of 0.55 ppm) was observed
for the 1′â hydroxy compounds 5a and 5b compared to the
epimer 5c, confirming the stereochemical assignments at C1′
and C2′.
reospecific reduction of the ketone) should similarly exhibit
NOEs to H24 of either a 24-O-methyl or MOM ether. The
reduced â-ketodihydropyrrole ring system, therefore, became
a stereochemical bridge between the two isolated zones.
The initial attempts at C24 O-methylation of selectively
protected derivative 1c, using several methods including boron
trifluoride diethyl etherate/diazomethane and trimethyloxonium
tetrafluoroborate, were unsuccessful. In fact, the reaction of
1c with the latter reagent exclusively afforded the elimination
product 4a. On the other hand, reaction of 1c with (MOM)Cl
gave the desired ether, 1d, in very good yield. The MOM group
tended to eliminate during NMR analysis in CDCl3 or CD2Cl2
to give 4a but was found to be stable in CD3CN. As would be
predicted, NaBH4 reduction of 1d gave the â-hydroxy compound
5a exclusively. The specificity for R-face reduction was
diminished in the absence of the MOM group, as evidenced by
the formation of a 9:1 mixture of epimeric alcohols 5b and 5c
during the sodium borohydride reduction of 1c.
Returning to 5a, H24 gave a NOESY correlation to H1′, albeit
weak (rH1′,H24, 3.3 Å), and to the MOM methylene group (Figure
5). Furthermore, the methylene protons also gave NOEs to the
1′-OH and H23. These observations are only possible with the
23â hydrogen, 24-â hydroxy, and 1′â hydroxy groups, thus
establishing the relative configuration at these centers. Very
weak NOESY correlations for both 5a and 5b were, once again,
observed between the anti protons H23 and H24, consistent with
the 149° dihedral angle and a 3.1 Å interatomic distance.
H1′ of both 5a and 5b gave strong NOESY correlations
(Figure 5) to H2′ (rH1′,H2′, 2.4 Å), thus indicating their cis
relationship. This was further substantiated by the Jvic ) 6.8
Hz, which is consistent with the measured dihedral angle (φ )
(c) Absolute Stereochemistry. The absolute stereochemistry
of nodulisporic acid A was determined by application of the
advanced Mosher method.8 Both the S- and R-MTPA esters
(1e and 1f) at C7 of the methyl ester 1b were prepared by

Studies on Nodulisporic Acid A Insecticide
J. Am. Chem. Soc., Vol. 119, No. 38, 1997 8813
Figure 6. ∆δ (δS - δR) values in ppm obtained for the R- and S-MTPA
esters of nodulisporic acid A methyl ester (1b).
seracata.9 Nodulisporic acid A is more active than paraher-
quamide10 (LC50 of 50 ppm in both assays) and less active than
ivermectin11 (LC50 of 0.02 and 0.045 ppb against A. aegypti
and L. seracata, respectively). For the comparison of the
biological activities, paxilline,3 one of the known indole
diterpenes, was tested in both assays and was found to be
inactive at 25 ppm in A. aegypti and at 250 ppm in L. seracata.
Conclusion
The structurally novel nodulisporic acid A is a potent
insecticide. It is more potent than paraherquamide and less
potent than the benchmark, ivermectin. The biological activity
does not seem to be a general feature of indole diterpenes, as
paxilline is inactive in the two assays tested. It bears some
structural resemblance to a number of previously identified
indole diterpenes, including janthitrem G,12 the shearinines,4 the
lolitrems,13 the penitrems,14 and the paspalitrems.15 All of these
compounds, except nodulisporic acid A, possess a tertiary
hydroxy group,12-15 a feature implicated in tremorgenic proper-
ties. One of the more interesting structural/biogenetic features
of nodulisporic acid A is the reversed ring fusion of the
dihydropyran compared to those janthitrems12 and shearinines4
and cyclopentyl rings in the western hemisphere of the molecule;
another is the unique, highly strained five-membered â-keto-
dihydropyrrole ring derived from isoprenylation of the indole
moiety. The latter unit is unprecedented in the indole
mycotoxins reported to date.
reaction with the R- and S-MTPA chlorides, respectively, in
methylene chloride. The 1H NMR spectra of both compounds
were carefully assigned using COSY, HMQC, and TOCSY
experiments and the chemical shift differences, ∆δ (δS - δR),
calculated. These differences were profound and are shown in
Figure 6. All protons to the right of the MTPA plane, which
dissects the molecule through C7-C4-C3-C14, have positive
∆δ increments, whereas negative values are observed for protons
on the other side of the plane. The magnitude of the shift
differences varies from 0.001 to 0.175 ppm, and significant long-
range shielding effects are detected as far away as H19. Even
though the 28-methyl appears to fall to the left of the MTPA
plane in the drawing, examination of a molecular model shows
it to reside just to the right of the plane, and this explains its
small positive ∆δ value. The clear division into positive and
negative increment regions leaves no ambiguity in the assign-
ment of the S configuration at C7, and, from this, the complete
absolute stereochemistry of nodulisporic acid A methyl ester
was inferred (Figure 6).
Experimental Section
Spectral Analysis. NMR spectra were determined on Varian Unity
500 and 400 spectrometers operating at 500 and 400 MHz for 1H and
at 125 and 100 MHz for 13C, respectively. 1H and 13C chemical shifts
are referenced to the solvent (CD2Cl2) signal at δ 5.32 (1H) and 53.8
(d) Solution Conformation. The NMR methods helped in
determination of the solution conformation of nodulisporic acid
A methyl ester (1b), which was identical to that of the solid
state conformation determined by X-ray crystallography of 1c.
The equatorial side chain at C8 stretches away from the core
of the molecule. The isopropenyl group at C2′ and the hydroxy
group at C24 tend to crowd the â-face of the â-ketodihydro-
pyrrole ring. The conformation readily accounts for the
exchange-broadened resonances of the isopropenyl group at C2′
as being due to restricted rotation about C2′-C3′. Supporting
evidence came from the 2′-epimeric compound 2, which was
obtained by treatment of 1b with DMAP in a mixture of CH3-
CN-CH2Cl2. The resonances were no longer exchange-
broadened in 2 as well as the oxidation products 3a and 3b,
suggesting relief of steric hinderance between the side chain
and groups on the â-face of the molecule, in particular H5â
and the 29-methyl.
1
ppm (13C). Homonuclear H-1H connectivities were determined by
using 2D COSY, double-quantum-filtered COSY, and 1D decoupled
experiments. Homonuclear 1H NOEs were obtained by 1D difference
NOE measurements and 2D NOESY/ROESY experiments. One-bond
1
heteronuclear H-13C connectivities were determined by 2D proton-
detected HMQC experiments and long-range 1H-13C connectivities by
(9) Oslind, D. A.; Felcetto, T.; Misura, A.; Ondeyka, J.; Smith, S.; Goetz,
M.; Shoop, W.; Mickle, W. Med. Vet. Entomol., submitted.
(10) (a) Ondeyka, J. G.; Goegelman, R. T.; Schaeffer, J. M.; Keleman,
L.; Zitano, L. J. Antibiot. 1990, 43, 1375. (b) Yamazaki, M. E.; Okuyama,
E.; Kobayashi, M.; Inoue, H. Tetrahedron Lett. 1981, 22, 135.
(11) Chabala, J. C.; Mrozik, H.; Tolman, R. L.; Eskola, P.; Lusi, A.;
Peterson, L. H.; Woods, M. F.; Fisher, M. H.; Campbell, W. C.; Egerton,
J. R.; Ostlind, D. A. J. Med. Chem. 1980, 23, 1134.
(12) de Jesus, A. E.; Steyn, P. S.; Van Heerden, F. R.; Vleggaar, R. J.
Chem. Soc. Perkin Trans. I 1984, 697.
(13) (a) Gallagher, R. T.; Hawkes, A. D.; Steyn, P. S.; Vleggaar, R. J.
Chem. Soc., Chem. Commun. 1984, 614. (b) Ede, R. M.; Miles, C. O.;
Meagher, L. P.; Munday, S. C.; Wilkins, A. L. J. Agric. Food. Chem. 1994,
42, 231 and references cited therein.
Biological Activity. Nodulisporic acid A has an LC50 of 0.5
ppm against A. aegypti and an LC50 of 0.3 ppm against L.
(14) (a) de Jesus, A. E.; Steyn, P. S.; Van Heerden, F. R.; Vleggaar, R.;
Wessels, P. L. J. Chem. Soc., Perkin Trans. I 1983, 1857 and references
cited therein.
(8) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem.
Soc. 1991, 113, 4092.
(15) Dorner, J. W.; Cole, R. J.; Cox, R. H.; Cunfer, B. M. J. Agric.
Food. Chem. 1984, 32, 1069 and references cited therein.

8814 J. Am. Chem. Soc., Vol. 119, No. 38, 1997
Ondeyka et al.
2D proton-detected HMBC experiments. Nalorac 3 mm triple-
resonance and dedicated 13C microprobes were used. Natural abundance
INADEQUATE spectra were obtained on ∼26 mg of 1a in experiments
H), 3.10 (br s, 24-OH), 3.40 (m, 7-H), 3.74 (s, 5′′-OCH3), 4.97 (br s,
4′-Hb), 5.10 (s, 2′-H), 5.21 (br s, 4′-Ha), 5.22 (d, J ) 6.4 Hz, 24-H),
5.89 (d, J ) 15.2 Hz, 1′′-H), 6.07 (d, J ) 3.2 Hz, 19-H), 6.40 (dd, J
) 15.2, 11.2 Hz, 2′′-H), 7.22 (d, J ) 11.2 Hz, 3′′-H), 7.71 (s, 16-H);
HRFAB-MS (m/z) 694.4172 (M + H; calcd for C44H56NO6, 694.4107).
7-(p-Bromobenzoyl)nodulisporic Acid A Methyl Ester (1c).
p-Bromobenzoyl chloride (16 mg, 0.07 mmol) was added to a cooled
(0 °C) solution of methyl ester 1b (34 mg, 0.05 mmol), diisopropyl-
ethylamine (0.1 mL), and 4-(dimethylamino)pyridine (DMAP, 10 mg)
in CH2Cl2 (0.5 mL). The solution was stirred at 0 °C for 10 min,
followed by stirring overnight at room temperature. Ice was added
after completion (TLC, hexane-EtOAc, 7:4) of the reaction, the whole
mixture was poured onto 50 mL of EtOAc, and the layers were
separated. The EtOAc layer was washed sequentially with 20 mL each
of water, 10% aqueous citric acid, water, 10% aqueous NaHCO3, and
water, dried, evaporated under reduced pressure, and chromatographed
by preparative TLC on silica gel to give pure benzoate 1c (38 mg,
88.4%) as a yellow powder. Crystallization from CH2Cl2-MeOH gave
thick yellow needles: mp 273-75 °C dec; [R]22D +82.5° (c 0.6, CHCl3);
IR (ZnSe) νmax 3527, 2976, 1713, 1636, 1591, 1455, 1378, 1271, 1246,
optimized for JCC values of 40 and 60 Hz using a 3 mm dedicated 13
C
microprobe, using the method of Bax and Mareci.16
Mass spectra, including high-resolution mass measurements, were
determined in the EI mode using a Ultramark 1960 standard. FAB-
MS (fast atom bombardment mass spectra) were recorded on a JEOL
HX110 mass spectrometer. UV spectra were measured in MeOH at
20 °C on a Beckman DU-20. Optical rotations were measured in
(CHCl3) at 22 °C at the sodium D line on a Perkin Elmer 241 instrument.
Isolation and Fermentation. Nodulisporium sp. MF5954 was
isolated as an endophytic fungus from an unidentified woody plant
tissue. The woody tissue was surface sterilized by sequential immersion
in dilute NaOCl solution followed by 95% ethanol. Branched conid-
iophores are nodulose, which distinguishes the genus from other similar
fungi, such as Geniculospororium, Xylocladium, and anamorphs of
Xylariales (Ascomycotina) such as Hypoxylon, Xylaria, Camillea, and
Biscogniauxia. These fungi often dwell inside woody plants. It might
be of interest to know that indole mycotoxins are unknown among fungi
of the Xylariales.17
1
1230, 1098, 1068, 1013, 919, 846, 755 cm-1; H NMR (400 MHz,
The Nodulisporium sp. was cultured at 25 °C in shake flasks from
vegetative mycelial growths on a rotary shaker for 21-28 days in a
nutrient medium. The nutrient medium consisted of glycerol (75 g/L),
glucose (10 g/L), ardamine pH (5 g/L), (NH4)2SO4 (2 g/L), soybean
meal (5 g/L), tomato paste (5 g/L), sodium citrate (2 g/L), and distilled
water at pH 7.0.
CDCl3) δ 0.98 (s, 28-H3), 1.14 (s, 33-H3), 1.19 (s, 30- or 29-H3), 1.27
(s, 29- or 30-H3), 1.34 (s, 32-H3), 1.35 (s, 31-H3), 1.44 (br s, 5′-H3),
1.48 (s, 34-H3), 1.51, 1.56, 1.75, 1.89-2.03 (m, 5-H2, 6-H2, 9H, 10-
H2, 11-H2), 1.80 (d, J ) 1.2 Hz, 6′′-H3), 2.32 (dd, J ) 14, 10.8 Hz,
13-HR), 2.74 (dd, J ) 14, 6.4 Hz, 13-Hâ), 2.85 (m, 12-H), 2.88 (dd,
J ) 6.0, 2.8 Hz, 23-H), 3.10 (br s, 24-OH), 3.70 (s, 5′′-OCH3), 4.92
(m, 7-H), 4.97 (br s, 4′-Hb), 5.06 (s, 2′-H), 5.17 (br s, 4′-Ha), 5.24 (d,
J ) 6.4 Hz, 24-H), 5.86 (d, J ) 15.6 Hz, 1′′-H), 6.05 (d, J ) 3.2 Hz,
19-H), 6.26 (dd, J ) 15.6, 11.2 Hz, 2′′-H), 7.11 (d, J ) 11.2 Hz, 3′′-
H), 7.55 (d, J ) 8.4 Hz, Ar-H2), 7.70 (s, 16-H), 7.80 (d, J ) 8.4 Hz,
Ar-H2); HRFAB-MS (m/z) 876.3439 (M + H; calcd for C51H58NO7-
Br79 + H, 876.3475).
Methyl ethyl ketone (MEK) extracts from 6 L of 24 day liquid shake
flask fermentations were filtered, combined, and evaporated to dryness
under vacuum to a weight of 8 g. This material was dissolved in CH2-
Cl2 (20 mL) and applied to a silica gel column (E. Merck silica gel 60,
4.5 cm × 100 cm, flow rate 6.5 mL/min) equilibrated in CH2Cl2-
MeOH (95:5). The column was washed with 9:1 CH2Cl2-MeOH
followed by 3:1 to elute 1a. The solvents were evaporated under
reduced pressure and crude 1a (200 mg) was charged to a Sephadex
LH20 column (Pharmacia, 2.5 cm × 200 cm, flow rate 5 mL/min) in
MeOH. Fractions containing 1a were combined (530-650 mL, 90
mg), dried under vacuum, and chromatographed further on an Eka
Nobel C-18 HPLC column (9.6 mm × 250 mm) using 70% CH3CN in
H2O containing 0.1% TFA to yield pure 1a (4.8 mg). The compound
had a Rf value of 0.47 on TLC plates (EM silica gel 60F254) with 9:1
CH2Cl2-MeOH. A similar process was used to purify material from
several further fermentation batches ranging in volumes from 1 to 20
L.
7-(p-Bromobenzoyl)-24-(methoxymethyl)nodulisporic Acid A Meth-
yl Ester (1d). To a cooled (-30 °C) solution of 1c (40 mg, 0.045
mmol) in CH2Cl2 (0.5 mL) was added diisopropylethylamine (0.5 mL)
and methoxymethyl chloride (0.3 mL), and the solution was stirred at
that temperature for 30 min, followed by overnight reaction at room
temperature. After completion of the reaction (large excess of reagents
needed), volatile reagents were removed under reduced pressure. The
product was purified directly on silica gel plates (hexane-EtOAc, 7:3),
and the yellow band was eluted with EtOAc. Evaporation of solvent
gave pure MOM ether 1d (30 mg) as an amorphous powder: IR (ZnSe)
ν
max 2975, 1718, 1591, 1455, 1366, 1268, 1231, 1098, 1034, 756 cm-1
;
Nodulisporic Acid A (1a): mp 250-255 °C; [R]22 +13° (c 0.4,
1H NMR (400 MHz, CDCl3) δ 0.98 (s, 28-H3), 1.06 (s, 33-H3), 1.20
(s, 30- or 29-H3), 1.27 (s, 29- or 30-H3), 1.33 (s, 32-H3), 1.35 (s, 31-
H3), 1.48 (br s, 5′-H3), 1.55 (s, 34-H3), 1.51, 1.58, 1.75, 1.89 -2.03
(m, 5-H2, 6-H2, 9H, 10-H2, 11-H2), 1.80 (d, J ) 1.2 Hz, 6′′-H3), 2.32
(dd, J ) 14, 10.8 Hz, 13-HR), 2.75 (dd, J ) 14, 6.4 Hz, 13-Hâ), 2.86
(m, 12-H), 3.00 (dd, J ) 4.4, 2.4 Hz, 23-H), 3.46 (s, (MOM)-OCH3),
3.71 (s, 5′′-OCH3), 4.86 (d, J ) 7.6 Hz, (MOM)-OCHb-O), 4.92 (m,
7-H), 4.95 (br s, 4′-Hb), 4.97 (s, 2′-H), 5.14 (br s, 4′-Ha), 5.15 (d, J )
4.4 Hz, 24-H), 5.32 (d, J ) 7.6 Hz, (MOM)-OCHa-O), 5.86 (d, J )
15.6 Hz, 1′′-H), 6.05 (d, J ) 2.8 Hz, 19-H), 6.26 (dd, J ) 15.6, 11.2
Hz, 2′′-H), 7.11 (d, J ) 10.8 Hz, 3′′-H), 7.55 (d, J ) 8.4 Hz, Ar-H2),
7.73 (s, 16-H), 7.80 (d, J ) 8.4 Hz, Ar-H2); HRFAB-MS (m/z) 920.3712
(M + H; calcd for C53H63NO8Br79, 920.3737).
D
CHCl3); UV (MeOH) λmax 241 (ꢀ ) 43 900), 265 (ꢀ ) 49 800), 385 (ꢀ
) 7360) nm; IR (ZnSe) νmax 2980, 2364, 1704 (br), 1378, 1228, 1034
cm-1; HRFAB-MS m/z 680.3891 (M + H; calcd for C43H53NO6 + H,
680.3951), 662.3790 (M + H - H2O; calcd for C43H51NO5 + H,
662.3845); 1H NMR (500 MHz, CD2Cl2), see Table 1; 13C NMR (125
MHz, CD2Cl2), see Table 1.
Nodulisporic Acid A Methyl Ester (1b). To a cooled (-78 °C)
solution of nodulisporic acid A (1a, 80 mg) in CH2Cl2 (1 mL) was
added an excess of an ethereal solution of CH2N2. Progress of the
reaction was monitored by TLC (CH2Cl2-MeOH, 9:1). The reaction
was complete within 5 min. Excess of CH2N2 was destroyed by
addition of HOAc, and solvents were removed to give clean methyl
ester (1b) as a yellow powder in quantitative yield. An analytical
sample of the methyl ester was prepared by chromatography on a small
silica gel column, eluting with 20-30% EtOAc in hexane. Crystal-
lization from CH2Cl2-MeOH gave yellow granules: mp 270-72 °C;
7-(S)-r-Methoxy-r-[(trifluoromethyl)phenylacetoyl]noduli-
sporic Acid A Methyl Ester (1e). A solution of (R)-(-)-R-methoxy-
R-(trifluoromethyl)phenylacetyl chloride (R-MTPACl, 25 mg) in 0.1
mL of CH2Cl2 was added to a solution of 1b (10 mg), pyridine (0.2
mL), and DMAP (25 mg) in 0.1 mL of CH2Cl2. The solution was
stirred at room temperature overnight. The progress of the reaction
was monitored by silica gel TLC (hexane-EtOAc, 3:2). Reaction was
extremely slow and was quenched with ice before disappearance of all
starting material. Fifty milliliters of EtOAc was added, and the organic
layer was washed with water, 10% aqueous citric acid, water, and 10%
aqueous NaHCO3, dried, and evaporated under reduced pressure.
Purification by preparative silica gel TLC gave pure (S)-MTPA ester
1e (3 mg) as an amorphous powder and unreacted 1b.
[R]22 + 19° (c 0.5, CHCl3); IR (ZnSe) νmax 3505, 2976, 1708, 1634,
D
1589, 1452, 1378, 1290, 1244, 1230, 1096, 1076, 1023, 1008, 980,
cm-1; 1H NMR (400 MHz, CD2Cl2) δ 0.97 (s, 28-H3), 1.06 (s, 30-H3),
1.13 (s, 33-H3), 1.15 (s, 29-H3), 1.32 (s, 32-H3), 1.34 (s, 31-H3), 1.43
(s, 34-H3), 1.48 (br s, 5′-H3), 1.48, 1.55 (m, 11-H2), 1.55 (m, 10-H2),
1.65 (m, 9-H), 1.76, 1.93 (m, 5-H2), 1.74, 1.78 (m, 6-H2), 1.96 (d, J )
1.2 Hz, 6′′-H3), 2.33 (dd, J ) 13.6, 10.8 Hz, 13-HR), 2.75 (dd, J )
13.6, 6.4 Hz, 13-Hâ), 2.82 (dd, J ) 6.4, 2.8 Hz, 23-H), 2.85 (m, 12-
(16) Bax, A.; Mareci, T. H. J. Magn. Reson. 1983, 53, 360.
(17) Whalley, A. J. S.; Edwards, R. L.Can. J. Bot. 1993, 73 (Suppl. 1),
S802.
1e: IR (ZnSe) νmax 3539, 2976, 1746, 1709, 1452, 1379, 1247, 1185,
1112, 1065, 1020, 722 cm-1; 1H NMR (400 MHz, CD2Cl2) (assignment

Studies on Nodulisporic Acid A Insecticide
J. Am. Chem. Soc., Vol. 119, No. 38, 1997 8815
is based on COSY, HMQC spectrum) δ 0.999 (s, 28-H3), 1.135 (s,
33-H3), 1.154 (s, 30-H3), 1.176 (s, 29-H3), 1.318 (s, 32-H3), 1.346 (s,
31-H3), 1.43 (s, 34-H3), 1.47 (br s, 5′-H3), 1.49 (m, 10-H2), 1.55, 1.77
(m, 11-H2), 1.746 (m, 9-H), 1.895, 1.989 (m, 5-H2), 1.90 (d, J ) 1.2
Hz, 6′′-H3), 1.93 (m, 6-H2), 2.34 (dd, J ) 14, 10.8 Hz, 13-HR), 2.769
(dd, J ) 14, 6.8 Hz, 13-Hâ), 2.82 (dd, J ) 6.4, 3.2 Hz, 23-H), 2.88
(m, 12-H), 3.47 (d, J ) 1.2 Hz, OCH3), 3.77 (s, 5′′-OCH3), 4.97 (br s,
4′-Hb), 4.98 (m, 7-H), 5.09 (s, 2′-H), 5.20 (br s, 4′-Ha), 5.229 (d, J )
6.4 Hz, 24-H), 5.931 (d, J ) 15.6 Hz, 1′′-H), 6.075 (d, J ) 3.2 Hz,
19-H), 6.3535 (dd, J ) 15.2, 11.2 Hz, 2′′-H), 7.24 (d, J ) 11.6 Hz,
3′′-H), 7.30-7.42 (m, Ar-H5), 7.722 (s, 16-H); HRFAB-MS (m/z) 910
(M + H; high-resolution data could not be measured due to low
intensity), 892.4335 [(M + H - H2O)+; calcd for C54H61NO7F3,
892.4399].
136.21 (C18), 138.33 (C25), 138.53 (C3′′), 139.81 (C3′), 153.17 (C1′′),
154.72 (C2), 160.75 (C27), 169.05 (C5′′), 198.06 (C1′); HRFAB-MS
(m/z) 694.4106 (M + H, calcd for C44H56NO6, 694.4107).
Oxidation Product 3a of Nodulisporic Acid A: 1H NMR (500
MHz, CD2Cl2) δ 1.08 (s, 30-H3), 1.13 (s, 34-H3), 1.15 (s, 28-H3), 1.30
(s, 32-H3), 1.34 (s, 31-H3), 1.41 (s, 33-H3), 1.45 (s, 29-H3), 1.90 (s,
5′-H3), 1.94 (d, J ) 1.2 Hz, 6′′-H3), 2.47 (dd, J ) 3.7, 11.8 Hz, 13-
HR), 2.81 (dd, J ) 3.0, 6.6 Hz, 23-H), 2.96 (m, 12-H), 3.31 (dd, J )
4.6, 11.2 Hz, 7-H), 3.56 (dd, J ) 5.7, 11.8 Hz, 13-Hâ), 5.03 (br s,
4′-Hb), 5.07 (br s, 4′-Ha), 5.14 (s, 2′-H), 5.20 (d, J ) 6.6 Hz, 24-H),
5.86 (d, J ) 15.4 Hz, 1′′-H), 6.20 (d, J ) 3.0 Hz, 19-H), 6.38 (dd, J
) 11.3, 15.4 Hz, 2′′-H), 7.28 (br d, J ) 11.3 Hz, 3′′-H), 8.28 (s, 16-
H); 13C NMR (125 MHz, CD2Cl2) δ 11.6 (C30), 12.6 (C6′′), 15.6 (C28),
19.1 (C29), 19.5 (C5′), 22.9 (C10), 23.3 (C34), 26.4 (C6), 27.5 (C11),
29.5 (C31), 29.9 (C33), 30.7 (C5), 31.5 (C32), 42.0 (C12), 44.2 (C9),
45.2 (C4), 45.4 (C13), 47.5 (C8), 56.0 (C3), 57.4 (C23), 72.7 (C20),
74.0 (C22), 75.5 (C2′), 75.5 (C24), 76.8 (C7), 112.5 (C4′), 123.3 (C26),
125.2 (C4′′), 126.3 (C2′′), 126.3 (C19), 128.7 (C16), 129.1 (C15), 133.9
(C17), 136.1 (C18), 139.9 (C3′), 140.7 (C3′′), 151.1 (C25), 154.2 (C1′′),
153.4 (C27), 172.4 (C5′′), 174.5 (C2), 197.8 (C14), 198.6 (C1′).
Oxidation Product 3b of Nodulisporic Acid A: 1H NMR (500
MHz, CD2Cl2) δ 1.04 (s, 30-H3), 1.13 (s, 34-H3), 1.15 (s, 28-H3), 1.30
(s, 32-H3), 1.34 (s, 31-H3), 1.41 (s, 33-H3), 1.44 (s, 29-H3), 1.90 (s,
5′-H3), 1.97 (d, J ) 1.2 Hz, 6′′-H3), 2.47 (dd, J ) 3.7, 11.8 Hz, 13-
HR), 2.81 (dd, J ) 3.0, 6.6 Hz, 23-H), 2.96 (m, 12-H), 3.27 (dd, J )
4.6, 11.2 Hz, 7-H), 3.56 (dd, J ) 5.7, 11.8 Hz, 13-Hâ), 5.03 (br s,
4′-Hb), 5.07 (br s, 4′-Ha), 5.14 (s, 2′-H), 5.20 (d, J ) 6.6 Hz, 24-H),
5.66 (d, J ) 15.4 Hz, 1′′-H), 6.20 (d, J ) 3.0 Hz, 19-H), 7.16 (dd, J
) 11.2, 15.4 Hz, 2′′-H), 6.56 (br d, J ) 11.2 Hz, 3′′-H), 8.28 (s, 16-
H).
7-(R)-r-Methoxy-r-[(trifluoromethyl)phenylacetoyl]noduli-
sporic Acid A Methyl Ester (1f). Prepared as described above from
(S)-(+)-MTPACl, to give a yellow amorphous powder: IR (ZnSe) νmax
3512, 2976, 1742, 1709, 1452, 1378, 1248, 1169, 1111, 1021, 721 cm-1
;
1H NMR (400 MHz, CD2Cl2) (assignment is based on COSY, HMQC
spectrum) δ 0.998 (s, 28-H3), 1.093 (s, 30-H3), 1.136 (s, 33-H3), 1.185
(s, 29-H3), 1.318 (s, 32-H3), 1.346 (s, 31-H3), 1.43 (s, 34-H3), 1.47 (br
s, 5′-H3), 1.473 (m, 10-H2), 1.55, 1.75 (m, 11-H2), 1.736 (m, 9-H),
1.855 (d, J ) 1.2 Hz, 6′′-H3), 1.91, 2.019 (m, 5-H2), 1.94 (m, 6-H2),
2.338 (dd, J ) 14, 10.8 Hz, 13-HR), 2.7645 (dd, J ) 14, 6.8 Hz, 13-
Hâ), 2.8185 (dd, J ) 6.4, 3.2 Hz, 23-H), 2.87 (m, 12-H), 3.48(d, J )
1.2 Hz, OCH3), 3.76 (s, 5′′-OCH3), 4.99 (br s, 4′-Hb), 5.0185 (m, 7-H),
5.098 (s, 2′-H), 5.22 (br s, 4′-Ha), 5.23 (d, J ) 6.4 Hz, 24-H), 5.821
(d, J ) 15.2 Hz, 1′′-H), 6.0755 (d, J ) 2.8 Hz, 19-H), 6.177 (dd, J )
15.2, 11.2 Hz, 2′′-H), 7.1215 (d, J ) 11.6 Hz, 3′′-H), 7.30-7.45 (m,
Ar-H5), 7.723 (s, 16-H), 13C NMR (100 MHz, CD2Cl2) δ 12.18 (C30),
12.80 (C6′′), 15.16 (C28), 19.49 (C29), 17.79 (C5′), 23.68 (C6), 24.25
(C10), 23.47 (C33), 25.55 (C11), 27.76 (C13), 29.91 (C31), 30.10
(C34), 31.97 (C32), 32.21 (C5), 39.02 (C4), 45.68 (C9), 46.01 (C8),
48.00 (C12), 51.99 (5′′-OCH3), 55.77 ((MTPA)-OCH3), 55.99 (C3),
58.22 (C23), 58.80 ((MTPA)-CF3), 71.44 ((MTPA)-C2), 72.59 (C20),
73.87 (C22), 75.34 (C24), 76.24 (C5′), 81.43 (C7), 113.5 (C26), 116.74
(C16), 117.57 (C4′), 121.73 (C15), 122.08 (C19), 122.83 (C14), 125.98
(C2′′), 126.69 (C4′′), 127.56 (2C, (MTPA)-Ar), 128.58 (2C, (MTPA)-
Ar), 129.83 ((MTPA)-Ar), 134.11 (C17), 135.90 (C18), 138.43 (C3′′),
138.50 (C25), 140.14 (C3′), 150.91 (C1′′), 154.26 (C2), 161.97 (C27),
165.94 ((MTPA)-C1), 168.97 (C5′′), 197.84 (C1′); HRFAB-MS (m/z)
910.4394 (low intensity, M + H; calcd for C54H63NO8F3, 910.4505),
892.4331 (M + H - H2O; calcd for C54H61NO7F3, 892.4399).
7-(p-Bromobenzoyl)-24-deoxy-23,24-dehydronodulisporic Acid A
Methyl Ester (4a). To a solution of 1c (28 mg, 0.032 mmol) in CH2-
Cl2 (0.7 mL) were added 1,8-bis(dimethylamino)naphthalene, N,N,N′,N′-
tetramethyl-1,8-naphthalenediamine (Proton Sponge, 30 mg, 0.14
mmol), and trimethyloxonium tetrafluoroborate (20 mg, 0.14 mmol),
and the solution was stirred overnight. A single, dark yellow, less polar
product was formed (TLC, hexane-EtOAc, 7:4). The crude reaction
mixture was directly charged onto silica gel preparative plates and
developed in the same solvent. The main band was eluted with EtOAc
to give 24 mg (87.5%) of pure 4a as a yellow powder: IR (ZnSe) νmax
2977, 1713, 1636, 1590, 1438, 1374, 1266, 1230, 1174, 1099, 1067,
1013, 850, 756 cm-1; 1H NMR (400 MHz, CDCl3) (only distinct shifts
have been listed) δ 0.97 (s, 28-H3), 1.20 (s, 3H), 1.28 (s, 3H), 1.46 (br
s, 3H), 1.49 (s, 6H), 1.56 (s, 3H), 1.57 (s, 3H), 1.80 (d, J ) 1.2 Hz,
6′′-H3), 2.31 (dd, J ) 14, 10.4 Hz, 13-HR), 2.74 (dd, J ) 14, 6.4 Hz,
13-Hâ), 2.78 (m, 12-H), 3.71 (s, 5′′-OCH3), 4.92 (m, 7-H), 4.93 (br s,
4′-Hb), 4.97 (s, 2′-H), 5.12 (br s, 4′-Ha), 5.87 (d, J ) 15.6 Hz, 1′′-H),
6.26 (dd, J ) 15.6, 11.2 Hz, 2′′-H), 6.76 (d, J ) 2.0 Hz, 19-H), 6.85
(dd, J ) 1.6, 0.4 Hz, 24-H), 7.11 (d, J ) 11.2 Hz, 3′′-H), 7.55 (d, J )
8.4 Hz, Ar-H2), 7.78 (s, 16-H), 7.81 (d, J ) 8.4 Hz, Ar-H2), HRFAB-
MS (m/z) 858.3398 (M + H, calcd for C51H57NO6Br, 858.3369).
7-(p-Bromobenzoyl)-24-(methoxymethyl)-1′â-hydroxynoduli-
sporic Acid A (5a). Sodium borohydride (10 mg) was added to a
solution of 1d (14 mg) in a mixture of THF (0.4 mL) and MeOH (0.4
mL) and stirred at room temperature. The reaction mixture almost
instantaneously became colorless, and the reaction was complete within
10 min. A single product was formed in the reaction (TLC, hexane-
EtOAc, 7:3). Acetone was added to consume excess reducing agent.
Solvents were removed under a stream of N2. The product was purified
on preparative silica gel plates (hexane-EtOAc, 7:3), and the band
was eluted with EtOAc, which gave 5a (10 mg) as a light yellow
powder: IR (ZnSe) νmax 3485, 2975, 1714, 1636, 1590, 1438, 1365,
C2′-(R)-Nodulisporic Acid A Methyl Ester (2). To a solution of
methyl ester 1b (20 mg) in CH3CN (4 mL) and CH2Cl2 (1 mL) was
added excess DMAP (80 mg). The solution was stirred at room
temperature for 48 h. TLC examination of the reaction mixture
indicated the formation of a less polar major product. Solvent was
removed from the reaction mixture, and the product was purified on a
preparative silica gel plate (hexane-EtOAc, 3:2). Two bands were
eluted with EtOAc. The more polar band gave 5.5 mg of unreacted
1b, and the less polar band afforded 9.9 mg of epimeric compound 2
as a yellow amorphous powder: [R]22D -57° (c 0.99, CHCl3); IR (ZnSe)
νmax 3511, 2974, 1707, 1635, 1587, 1459, 1378, 1289, 1244, 1229,
1
1077, 1023, 980, cm-1; H NMR (400 MHz, CD2Cl2) (assignments
based on COSY, HMQC, and HMBC experiments) δ 1.06 (s, 30-H3),
1.09 (s, 29-H3), 1.148 (s, 28-H3), 1.154 (s, 33-H3), 1.32 (s, 32-H3),
1.35 (s, 31-H3), 1.43 (s, 34-H3), 1.47 (m, 10-H2), 1.57, 1.78 (m, 5-H2),
1.74 (m, 9-H), 1.76, 1.78 (m, 6-H2), 1.60, 1.76 (m, 11-H2), 1.79 (s,
5′-H3), 1.96 (d, J ) 1.2 Hz, 6′′-H3), 2.43 (dd, J ) 13.6, 10.8 Hz, 13-
HR), 2.71 (dd, J ) 13.6, 6.8 Hz, 13-Hâ), 2.78 (dd, J ) 6.4, 2.8 Hz,
23-H), 2.80 (m, 12-H), 3.38 (m, 7-H), 3.74 (s, 5′′-OCH3), 4.88 (s, 4′-
Hb), 5.03 (s, 2′-H), 5.17 (apparent t, 4′-Ha), 5.25 (d, J ) 6.0 Hz, 24-
H), 5.88 (d, J ) 15.6 Hz, 1′′-H), 6.05 (d, J ) 2.8 Hz, 19-H), 6.40 (dd,
J ) 15.6, 11.2 Hz, 2′′-H), 7.22 (d, J ) 11.2 Hz, 3′′-H), 7.71 (s, 16-H);
13C NMR (100 MHz, CD2Cl2) δ 11.15 (C30), 12.95 (C6′′), 14.12 (C28),
19.28 (C5′), 19.74 (C29), 23.55 (C33), 24.96 (C10), 25.71 (C11), 26.14
(C6), 27.64 (C13), 29.91 (C31), 30.13 (C34), 31.98 (C32), 33.97 (C5),
39.24 (C4), 44.92 (C9), 47.68 (C8), 49.93 (C12), 51.98 (5′′OCH3), 54.73
(C3), 58.19 (C23), 72.58 (C20), 73.86 (C22), 75.38 (C24), 75.64 (C2),
76.62 (C7), 112.96 (C26), 115.25 (C4′), 116.74 (C16), 121.41 (C15),
121.76 (C14), 122.07 (C19), 126.02 (C2′′), 126.15 (C4′′), 134.46 (C17),
1
1268, 1246, 1100, 1013, 756 cm-1; H NMR (400 MHz, CD3CN) δ
0.97 (s, 28-H3), 1.06 (s, 33-H3), 1.16 (br s, 29-H3), 1.27 (s, 30-H3),
1.28 (s, 31-H3, 32-H3), 1.42 (s, 34-H3), 1.44, 1.51 (m, 10-H2), 1.53,
1.74 (m, 11-H2), 1.73 (d, J ) 1.2 Hz, 6′′-H3), 1.86 (m, 9H), 1.92 (m,
6-H2), 1.92 (br s, 5′-H3), 1.93, 2.10 (m, 5-H2), 2.23 (dd, J ) 14, 11
Hz, 13-HR), 2.65 (dd, J ) 14, 6.4 Hz, 13-Hâ), 2.77 (m, 12-H), 2.88
(dd, J ) 6.0, 3.0 Hz, 23-H), 3.39 (s, (MOM)-OCH3), 3.65 (s, 5′′-
OCH3), 4.20 (br s, 1′-OH), 4.86 (s, (MOM)-OCH2-O), 4.92 (br s,
4′-Hb), 4.95 (m, 7-H), 5.06 (br s, 4′-Ha), 5.13 (br d, J ) 5.4 Hz, 24-
H), 5.32 (very broad s, 2′-H), 5.99 (d, J ) 15.4 Hz, 1′′-H), 6.04 (d, J

8816 J. Am. Chem. Soc., Vol. 119, No. 38, 1997
Ondeyka et al.
) 2.9 Hz, 19-H), 6.20 (br t, J ) 7.0 Hz, 1′-H, collapsed to a doublet,
J23-24H) 6.8 Hz, after decoupling at δ 4.20, and a broad doublet after
decoupling at 5.33 ppm), 6.33 (dd, J ) 15.64, 11.2 Hz, 2′′-H), 7.02 (d,
J ) 11.1 Hz, 3′′-H), 7.37 (s, 16-H), 7.64 (d, J ) 8.8 Hz, Ar-H2), 7.84
(d, J ) 8.8 Hz, Ar-H2); HRFAB-MS (m/z) 922.3890 (M + H; calcd
for C53H65NO8Br, 922.3893).
5c: IR (ZnSe) νmax 3448, 2972, 1713, 1636, 1591, 1438, 1375, 1268,
1247, 1101, 1068, 1012, 846, 756 cm-1; 1H NMR (400 MHz, CD3CN)
δ 1.09 (s, 28-H3), 1.14 (s, 33-H3), 1.17 (br s, 29-H3), 1.26 (s, 31-H3),
1.29 (s, 30-H3), 1.30 (s, 34-H3, 32-H3), 1.38 (s, (br s, 5′-H3),), 1.45,
1.50 (m, 10-H2), 1.55, 1.75 (m, 11-H2), 1.65 (m, 9H), 1.73 (d, J ) 1.2
Hz, 6′′-H3), 1.93 (m, 6-H2), 1.95-2.05 (m, 5-H2), 2.38 (dd, J ) 13.6,
11.2 Hz, 13-Hb), 2.59 (dd, J ) 13.6, 6.8 Hz, 13-Ha), 2.64 (dd, J )
5.6, 2.8 Hz, 23-H), 2.75 (m, 12-H), 3.33 (d, J ) 7 Hz, 24-OH), 3.65
(s, 5′′-OCH3), 4.66 (broad hump, 4′-Hb), 4.91 (dd, J ) 12, 4.4 Hz,
7-H), 5.04 (t, J ) 6.8 Hz, 24-H), 5.08 (br s, 4′-Ha), 5.29 (broad hump,
2′-H, sharpened after decoupling δ 6.25 ppm), 5.98 (d, J ) 15.6 Hz,
1′′-H), 5.99 (d, J ) 2.8 Hz, 19-H), 6.25 (d, J ) 2.8 Hz, 1′-H), 6.34
(dd, J ) 15.6, 11.2 Hz, 2′′-H), 7.02 (d, J ) 11.2 Hz, 3′′-H), 7.30 (s,
16-H), 7.64 (d, J ) 8.8 Hz, Ar-H2), 7.84 (d, J ) 8.8 Hz, Ar-H2);
HRFAB-MS (m/z) 878.3582 (M + H, calcd for C51H61NO7Br,
878.3631).
7-(p-Bromobenzoyl)-1′â- and -1′r-hydroxynodulisporic Acid A
(5b and 5c). Sodium borohydride reduction of 1c (14 mg) in a manner
similar to that described above, followed by purification by preparative
TLC, gave 11 mg of 5b and 1.1 mg of 5c, both as amorphous powders.
5b: IR (ZnSe) νmax 3448, 2975, 1713, 1591, 1437, 1365, 1269, 1247,
1
1099, 1067, 1013, 993, 756 cm-1; H NMR (400 MHz, CD3CN) δ
1.00 (s, 28-H3), 1.11 (s, 33-H3), 1.13 (br s, 29-H3), 1.26 (s, 31-H3),
1.28 (s, 30-H3), 1.30 (s, 32-H3), 1.37 (s, 34-H3), 1.45, 1.50 (m, 10-H2),
1.53, 1.72 (m, 11-H2), 1.73 (d, J ) 1.2 Hz, 6′′-H3), 1.86 (m, 9H), 1.93
(m, 6-H2), 1.93 (br s, 5′-H3), 1.93, 2.10 (m, 5-H2), 2.23 (dd, J ) 14,
10.8 Hz, 13-HR), 2.639 (dd, J ) 5.6, 2.8 Hz, 23-H), 2.643 (dd, J )
13.6, 10.8 Hz, 13-Hâ), 2.75 (m, 12-H), 3.65 (s, 5′′-OCH3), 4.00 (br s,
24-OH), 4.50 (br s, 1′-OH), 4.94 (m, 7-H), 4.95 (br s, 4′-Hb), 4.97
(apparent t, J ) 1.6 Hz, 4′-Ha), 5.13 (dd, J ) 5.6, 2.8 Hz, 24-H), 5.34
(very broad s, 2′-H, sharpened after decoupling δ 6.29 ppm), 5.99 (d,
J ) 15.6 Hz, 1′′-H), 6.04 (d, J ) 3.2 Hz, 19-H), 6.29 (br dd, J ) 6.8,
4.4 Hz, 1′-H, collapsed to a doublet, J23-24H ) 6.4 Hz, after decoupling
at δ 4.50 ppm and a broad doublet after decoupling at δ 5.33 ppm),
6.33 (dd, J ) 15.6, 11.2 Hz, 2′′-H), 7.02 (d, J ) 11.6 Hz, 3′′-H), 7.32
(s, 16-H), 7.64 (d, J ) 8.8 Hz, Ar-H2), 7.84 (d, J ) 8.8 Hz, Ar-H2);
13C NMR (400 MHz, CD3CN) δ 12.71 (C6′′), 12.79 (C30), 15.39 (C28),
19.74 (C29), 23.53 (C33), 24.15 (C6), 24.18 (very weak in broad-band
and DEPT spectrum due to exchange broadening, assigned by HMQC,
C5′), 24.88 (C10), 26.09 (C11), 28.41 (C13), 30.19 (C32), 30.42 (C34),
32.21 (C31), 32.69 (C5), 39.55 (C4), 46.14 (C8), 46.93 (C9), 47.94
(C12), 52.19 (5′′-OCH3), 56.2 (C3), 59.52 (C23), 72.95 (C20), 74.28
(C22), 75.44 (C24), 76.45 (C2′ by HMQC only), 79.05 (C1′ by HMQC
only), 80.10 (C7), 109.65 (C16), 112.32 (C26), 117.63 (C4′′ by HMQC
only), 120.81 (C19), 125.60 (C2′′), 126.39 (C4′′), 128.30, 130.30,
131.92, 132.73 (all benzoate), 134.31 (C17), 137.57 (C25), 139.03
(C3′′), 152.64 (C2), 153.17 (C1′′), 160.77 (C27), 165.70 (benzoate),
169.25 (C5′′) (signals for C14, C15, and C18 were not detected,
probably due to exchange broadening); HRFAB-MS (m/z) 878.3635
(M + H, calcd for C51H61NO7Br, 878.3631).
7-(p-Bromobenzoyl)-23,24-dehydro-24-deoxy-1′â-hydroxy-
nodulisporic Acid A (4b). When dissolved in CDCl3 (and even, on
occasion, in CD2Cl2), 5a tended to give the elimination product 4b as
a yellow powder: IR (ZnSe) νmax 3488, 2976, 1713, 1636, 1591, 1435,
1
1373, 1265, 1230, 1173, 1099, 1067, 1012, 993, 847, 756 cm-1; H
NMR (400 MHz, CD2Cl2) (only distinct shifts listed) δ 0.99 (br s, 3H),
1.17 (br s, 3H), 1.29 (s, 3H), 1.45 (br s, 6H), 1.53 (s, 9H), 1.79 (d, J
) 1.6 Hz, 6′′-H3), 2.25 (dd, J ) 13.2, 11.6 Hz, 13-Hb), 2.69 (dd, J )
13.6, 6.4 Hz, 13-Ha), 2.77 (m, 12-H), 3.68 (s, 5′′-OCH3), 4.02 (broad
hump, 1′-OH), 4.95 (m, 7-H), 5.10 (br s, 4′-Hb), 5.27 (broad hump,
2′-H), 5.91 (d, J ) 15.2 Hz, 1′′-H), 6.30 (dd, J ) 15.6, 11.2 Hz, 2′′-
H), 6.55 (d, J ) 1.6 Hz, 19-H), 6.58 (dd, J ) 1.6 Hz, 24-H), 7.10 (d,
J ) 11.2 Hz, 3′′-H), 7.47 (s, 16-H), 7.59 (d, J ) 8.8 Hz, Ar-H2), 7.84
(d, J ) 8.8 Hz, Ar-H2); HRFAB-MS (m/z) 860.3472 (M + H, calcd
for C51H59NO6Br, 860.3526).
Acknowledgment. We thank Gabe Dezeny for fermentation
support and Kevin Byrne for furnishing 13C-labeled nodulisporic
acid A.
JA971664K