Studies on paraherquamide biosynthesis: synthesis of deuterium-labeled 7-hydroxy-pre-paraherquamide, a putative precursor of paraherquamides A, E, and F

Article abstract of DOI:10.1016/j.tet.2008.08.102

The stereocontrolled, asymmetric synthesis of triply deuterium-labeled 7-hydroxy-pre-paraherquamide (27) was accomplished, employing a diastereoselective intramolecular S_N2′ cyclization strategy. The deuterium-labeled substrate was interrogated in a precursor incorporation experiment in the paraherquamide-producing organism Penicillium fellutanum. The isolated sample of paraherquamide A revealed incorporation of one of the two geminal deuterons of the CD₂-group at C-12 exclusively. The lack of signals for the second deuteron of the CD₂-group at C-12 and for the CH₂D-group (C-22/C-23) suggests that this substrate suffered an unexpectedly selective catabolic degradation and metabolic re-incorporation of deuterium thus casting doubt on the proposed biosynthetic intermediacy of 27. Consideration of alternative biosynthetic pathways, including oxidation of the indole C-6 position prior to hydroxylation at C-7 or oxidative spiro-contraction of pre-paraherquamide prior to construction of the dioxepin is discussed. The synthesis of 27 also provides for a concise, asymmetric stereocontrolled synthesis of an advanced intermediate that will be potentially useful in the synthesis of paraherquamides E and F.

Full text of DOI:10.1016/j.tet.2008.08.102

Tetrahedron 65 (2009) 3246–3260
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Studies on paraherquamide biosynthesis: synthesis of deuterium-labeled
7-hydroxy-pre-paraherquamide, a putative precursor
of paraherquamides A, E, and F
,b,
Konrad Sommer a, Robert M. Williams a
*
a Department of Chemistry, Colorado State University, Fort Collins, CO 80523, United States
b University of Colorado Cancer Center, Aurora, CO 80045, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 May 2008
Received in revised form 19 August 2008
Accepted 20 August 2008
Available online 11 September 2008
The stereocontrolled, asymmetric synthesis of triply deuterium-labeled 7-hydroxy-pre-paraherquamide
(27) was accomplished, employing a diastereoselective intramolecular SN20 cyclization strategy. The
deuterium-labeled substrate was interrogated in
a precursor incorporation experiment in the
paraherquamide-producing organism Penicillium fellutanum. The isolated sample of paraherquamide A
revealed incorporation of one of the two geminal deuterons of the CD2-group at C-12 exclusively. The
lack of signals for the second deuteron of the CD2-group at C-12 and for the CH2D-group (C-22/C-23)
suggests that this substrate suffered an unexpectedly selective catabolic degradation and metabolic re-
incorporation of deuterium thus casting doubt on the proposed biosynthetic intermediacy of 27.
Consideration of alternative biosynthetic pathways, including oxidation of the indole C-6 position prior
to hydroxylation at C-7 or oxidative spiro-contraction of pre-paraherquamide prior to construction of the
dioxepin is discussed. The synthesis of 27 also provides for a concise, asymmetric stereocontrolled
synthesis of an advanced intermediate that will be potentially useful in the synthesis of paraherquamides
E and F.
Keywords:
b-Methylproline
Asymmetric synthesis
Paraherquamide
Asperparaline
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
B (9),11 which contain a spiro-indoxyl rather than a spiro-oxindole,
and the asperparalines, which contain a spiro-succinimide,12 are
The paraherquamides constitute part of an unusual family of
prenylated indolic natural products derived from various genera of
fungi, which contain a common bicyclo[2.2.2]diazaoctane core
structure, a spiro-oxindole, and a substituted proline moiety. The
parent member, paraherquamide A 1, wasfirst isolated fromcultures
of Penicillium paraherquei by Yamazaki and co-workers in 1981.1
Since then, paraherquamides B–I,2 VM55595, VM55596, and
VM55597,3 SB203105 and SB200437,4 and sclerotiamide5 have been
isolated from various Penicillium and Aspergillus species. Marcfor-
tines A–C are structurally similar, containing a pipecolic acid unit in
place of proline.6 In addition, VM55599,3 aspergamides A and B,7
avrainvillamide (CJ-17,665),8 and the most recently isolated mem-
bers of this family, stephacidin A (10), stephacidin B (11),9 and
malbrancheamide10 are closely related members of this rapidly
expanding family of prenylated indole alkaloids. This last group of
compounds contains a 2,3-disubstituted indole in place of the spiro-
oxindole, spiro-indoxyl, or spiro-succinimide. Brevianamides A and
also structurally related (Fig. 1).
The members of this family of prenylated alkaloids have
attracted considerable attention due to their molecular complexity,
intriguing biogenesis, and biological activities.13 Some members,
most notably paraherquamide A, display potent anthelmintic
activity and antinematodal properties.14 In this context, since the
paraherquamides are unique both structurally and in their mode of
action,15 they represent a potentially promising new class of an-
thelmintics. Accordingly, modifications of paraherquamide A led to
the discovery of 2-deoxoparaherquamide A (PNU-141962), which
exhibits comparable biological activities to that as paraherquamide
A, but has an improved safety profile and is currently undergoing
evaluation in clinical studies.16 Stephacidins A (10) and B (11)9 have
also displayed promising anti-cancer activity through a unique
mechanism of action that is just beginning to emerge.17 Of partic-
ular interest was the report that stephacidin B exhibited selective,
antitumor activity against a testosterone-sensitive prostrate LNCaP
cell line with an IC50 value of 0.06 m
M.9
From a biogenetic perspective, the paraherquamides along
with the brevianamides, asperparalines, marcfortines, the mal-
brancheamides, notoamides18 and sclerotiamide comprise an
* Corresponding author. Tel.: þ1 970 491 6747; fax: þ1 970 491 3944.
E-mail address: rmw@lamar.colostate.edu (R.M. Williams).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.08.102

K. Sommer, R.M. Williams / Tetrahedron 65 (2009) 3246–3260
3247
labeled compounds 20–23.22 These experiments revealed that
only intermediate 20 was incorporated into paraherquamide A,
while racemic, doubly 13C-labeled VM55599 (22), 23, and 21
were not incorporated. Significantly, pre-paraherquamide
23 O
Me
22
1
NH
O
Me
2
Me
19H
21
3
24
Me
H
8
NMe
O
R
13 29Me20
17
Me
9
7
6
25
H
O
10
11
O
14
M2e7
O 26
Me 28
Me
N
N
4
O
N18 12
5
Me
15
has been detected as
a natural metabolite of the para-
N
16
8, asperparaline A
1, paraherquamide A, R = OH
2, paraherquamide E, R = H
herquamide-producing organism P. fellutanum and the asper-
paraline-producing organism Aspergillus japonicus.23 The lack of
incorporation of the dioxopiperazine 21 raises interesting ques-
tions concerning the timing of the reduction of the tryptophan-
derived carbonyl group. The incorporation of 20 indicates that
formation of the bicyclo[2.2.2]diazaoctane occurs at the stage of
the non-oxidized tryptophyl moiety. This mandates that oxida-
tions of the indole ring to form both the catechol-derived
dioxepin and spiro-oxindole must occur after the formation of
pre-paraherquamide. The dioxepin-derived isoprenylation and
the S-adenosylmethionine-mediated N-methylation reactions
probably occur late in the pathway.
H
Me
Me
H
N
O
Me
Me
H
Me
N
NH
H
O
O
R
N
N
O
Me
Me
Me
9, (-)-brevianamide B
O
O
N
Me
3, paraherquamide F (VM55594), R = H
4, paraherquamide G, R = OH
Me
H
Me
O
O
N
Me
H
24
O
1
23
Me
Me
2
NH
3
H
N
18 H
25
O
N
8
22
9
7
26
H19
10
17
Me28
Me29
16
15
10, (+)-stephacidin A
O 21
11
O27
12HO 4
O
6
20N
Me
N
5
13
14
OH
For asperparaline A, in addition sharing the bicyclo[2.2.2]di-
azaoctane core, the orientation of the spiro-succinimide ring is
consistent with the spiro-ring configuration of the para-
herquamides. It has been demonstrated that in paraherquamide A
Me
Me
Me
H
N
O
5, (-)-sclerotiamide
O
Me
Me
H
O
O
Me
H
NH
Me
N
O
O
O
N
biosynthesis,
from
L-tryptophan, L-b-methylproline (which is derived
O
R
O
H
N
+
O
L
-isoleucine), and an isoprene moiety are coupled together
N
N
Me
H
O
Me
Me
N
to provide the hexacyclic indole 20.24 Based on the experimental
Me
observation that the same three primary biosynthetic building
blocks (L-tryptophan, L-isoleucine, and dimethylallyl pyrophos-
N
6, marcfortine A, R = Me
7, marcfortine B, R = H
11, (-)-stephacidin B
phate) constitute asperparaline A, we proposed a unified bio-
genesis of the paraherquamides and the asperparalines as shown
Figure 1. Structures of selected paraherquamides and several related prenylated in-
dole alkaloids.
in Scheme 2.25 As previously reported, coupling of
L-tryptophan,
L-b-methylproline, and dimethylallyl pyrophosphate (DMAPP)
gives compound 20 via intramolecular Diels–Alder cycloaddition,
which can be oxidized to the hypothetical intermediate 24.
Compound 24 is considered to be a key branch-point species for
the two respective pathways to paraherquamide A and asper-
paraline A. In paraherquamide biosynthesis, 24 would suffer
prenylation and conversion to the dioxepin. In asperparaline
biosynthesis, this compound would undergo further oxidation
ultimately removing four carbon atoms from the benzenoid ring
of the tryptophan moiety.
In order to test this hypothesis experimentally and to further
penetrate the biogenesis of paraherquamide A and asperparaline A,
we have initiated studies on the synthesis of 2H- and/or 13C-labeled
hexacyclic putative biosynthetic intermediates 26–29 that will be
useful to investigate the intermediate stages in the biosynthesis of
paraherquamide A and asperparaline A.
interesting class of structurally related secondary metabolites
containing a bicyclo[2.2.2]diazaoctane core.
An emerging body of evidence supports the notion that this
structural motif is formed by a biosynthetic intramolecular [4þ2]
cycloaddition of the isoprene-derived olefin across a preformed
azadiene moiety derived from an oxidized piperazinedione (12/13)
as shown in Scheme 1.13,19,20
Me
Me
Me
Me
Me
H
Me
H
OH
N
HO
H
N
O
N
N
N
N
X
X
X
12
13
14
We are especially interested in the exact sequence of hydroxy-
lations and prenylation of the indole nucleus to fashion the unique
dioxepin moiety of paraherquamide A and the related dihydropyran
system of paraherquamides F and G. On the other hand, the oxi-
dative construction of the unique spiro-succinimide ring system of
asperparaline A from the tryptophan residue provides a challenging
and mechanistically provocative biosynthetic puzzle.
Recent interest in identifying new structural classes of anthel-
mintics is due to the appearance of drug resistance, and thus, the
unique mode of action of the paraherquamides and their consid-
erable biological activity have provided the motivation for us to
contemplate total syntheses of paraherquamides E (2), F (3) and the
asperparalines that would be readily adaptable to the preparation
of analogues.
Scheme 1. Proposed Diels–Alder reaction forming the core structure of brevianamides,
paraherquamides, and asperparalines.
In this context, Everett and co-workers described in 1993 the
isolation of VM55599 (21, Scheme 2), a minor metabolite from
culture extracts of a Penicillium sp. (IMI332995) that also pro-
duces paraherquamide A (1), among other paraherquamides.3
Taking into account the structural similarities between these
co-occurring metabolites, these authors proposed that VM55599
might indeed be a biosynthetic precursor of paraherquamide A.3
However, recent experimental observations from this laboratory
brought into question the capacity of VM55599 to serve as
a biosynthetic precursor to the paraherquamides and we pro-
posed a distinct, unified biogenesis of the paraherquamides and
VM55599.21 In this proposal, the biosynthetic precursors of the
paraherquamides and that of VM55599 would arise as di-
astereomeric products of the Diels–Alder cycloaddition of
a common azadiene through two of four possible diastereomeric
transition states. This hypothesis was recently experimentally
tested through feeding experiments of racemic, doubly 13C-
Our synthetic strategy to access these
b-methylproline-con-
taining natural products entailed an oxidative spiro-ring contrac-
tion of the generic hexacycle 30 that would be constructed via
a face-selective intramolecular SN20 cyclization reaction (Scheme
3).26 The 3-hydroxy analogue of compound 32 has been prepared
for the asymmetric total synthesis of paraherquamide A and has
been deployed for the synthesis of a hexacyclic species analogous to

3248
K. Sommer, R.M. Williams / Tetrahedron 65 (2009) 3246–3260
H
H
N
Me
Me
X
N
Me
O
O
Me
HO
H
OH
Me
Me
H
OH
-2H2O
[4e ox]
OH
N
Me
H
NH2
16, L-Trp
NH2
N
H
-
N
Me
15, L-Ile
N
19
+
N
Me
Me
OPP
X
18
[4+2]
17, DMAPP
X
H
N
H
H
N
Me
Me
H
Me
Me
N
OH
Me
H
Me
[ox]
H
H
Me
H
H
O
H
H
O
O
Me
Me
H
N
N
N
N
24
SAM; [ox]
N
N
[ox]
MAJOR
MINOR
X
X
OPP
Me
Me
20, X = H2
21, X = O
R
22, X =H ,
2
23, X = O
VM55599
O
O
Me
Me
Me
OH
N
Me
H
NH
NMe
Me
H
Me
H
O
O
OH
H
H
O
Me
N
O
N
Z
O
Me
Me
N
Me
O
R
N
Me
Me
Me
N
N
25
1, paraherquamide A & congeners
8, asperparaline A
Scheme 2. Proposed unified biogenesis of paraherquamides and asperparalines.
X
H
N
offers a rapid, versatile, and inexpensive access to a range of further
putative biosynthesis intermediates and inherently permits the
incorporation of 13C-labeling strategies as well. Moreover, this
approach should prove to be a useful strategy by which para-
herquamides E and F as well as asperparaline A might be
constructed.
Me
Y
Me
H
H
2, 3
Cl
Me
O
H
Me
N
N
30
TBSO
Me
O
Y
Me
O
X
Me
NH
2. Results and discussion
OEt
NBoc
32
N
NBoc
O
31
We recently described two practical methods for the synthesis
of the optically active a-alkylated-b-methylproline species 32. With
Scheme 3. Retrosynthetic approach to paraherquamides E and F.
this key substance in hand and in light of the required regiose-
lective transformations of the C-6/C-7-dihydroxyindole moiety at
the late stage of the synthesis of 26–29, we desired a convenient
synthesis of the differentially protected tryptophan derivative 44
(Scheme 4). Accordingly, starting from vanillin (33), acetylation
followed by nitration30 gave the 3-nitro-isomer exclusively, which
upon basic hydrolysis of the acetate and subsequent O-benzylation
afforded 37. Henry reaction and subsequent dehydration yielded
the dinitrostyrene 38.31 Reductive cyclization of 38 provided the
expected indole 39. Functionalization of the indole 3-position was
carried out by treatment of 39 with formaldehyde/dimethylamine
to give the corresponding gramine derivative 40, which was then
utilized in a Somei–Kametani-type reaction32 to access tryptophan
derivative 41. Finally, cleavage of the benzophenone imine group
and subsequent Boc-protection followed by basic hydrolysis affor-
ded the desired tryptophan derivative 44.
In our previous work on the total synthesis of paraherquamides
A and B, the synthetic strategy deployed to reach substrates anal-
ogous to 46 included a stepwise formation of the dioxopiperazine
ring, two methoxy carbonylations followed by a Somei–Kametani
coupling and decarboxylation.33 In an effort to improve upon this
protocol, a new and more efficient strategy that exploited two
successive peptide couplings was investigated.34,35
30.27 Our plan thus required the synthesis of the critical 2-
substituted, 3-methylproline derivative (32) with the desired
absolute and relative stereochemistry. Conversion of 32 to species
31 has been well documented in our laboratory as well as the
subsequent intramolecular SN20 cyclization, and Wacker-type
construction of the 2,3-disubstituted indole (30).28 Oxidative spiro-
ring contraction would then be expected to provide entry to both
paraherquamides E and F.29 The major advantage of this strategy is
that at the same time all four candidate precursors 26–29 (Fig. 2)
are accessible from a single synthesis. In addition, both of the 13C
labels in these compounds would be derived from relatively in-
expensive 13C-glycine and 1-13C-ethyl bromo-acetate.
R1
H
N
Me
2HCH2
H
R2
H
OR3
Me
N
2H
=
13C
N
2H
26: R1 = OCH2CH=CMe2, R2 = OH, R3 = Me
27: R1 = OH, R2 = H, R3 = H
28: R1 = H, R2 = OH, R3 = H
Accordingly, coupling of the tryptophan derivative 44 with
methylproline fragment 32 was accomplished with the pyBOP (or
BOP) reagent (Scheme 5). The subsequent selective removal of the
Boc protecting group in the presence of the acid-labile O-TBS pro-
tecting group afforded a w2:1 mixture of syn/anti diastereomers,
which were separated by column chromatography. Cyclization
assisted by 2-hydroxypyridine gave the dioxopiperazine 46. Start-
ing from the anti-dioxopiperazine 46b, protection of the secondary
amide as the corresponding lactim ether was accomplished
29: R1 = H, R2 = H, R3 = Me
Figure 2. Synthesis targets for biosynthesis studies.
We report here, a preparative synthesis of D3-7-hydroxy-pre-
paraherquamide (27) that has subsequently been used in a feeding
experiment with P. fellutanum. Significantly, this synthetic strategy

K. Sommer, R.M. Williams / Tetrahedron 65 (2009) 3246–3260
3249
CHO
CHO
CHO
NO2
c
a
b
HO
AcO
AcO
OMe
33
OMe
34
OMe
35
CHO
CHO
NO2
g
BnO
NO2
NO2
e, f
HO
d
NO2
BnO
OMe
OMe
OMe
38
36
37
NMe2
i
O
N
OEt
h
Ph
Ph
N
H
BnO
N
H
BnO
OMe
OMe
40
N
H
BnO
OEt
39
41
OMe
O
O
O
OEt
NH2
OH
NHBoc
j
l
k
NHBoc
N
H
BnO
N
H
N
BnO
BnO
H
OMe
OMe
OMe
42
43
44
Scheme 4. (a) Ac2O, DMAP, Et2O, 20 h, room temperature, 96%; (b) HNO3 fuming, ꢀ20 ꢁC, 3 h; (c) NaOH, H2O, room temperature, 20 min, 72–76% (two steps); (d) BnBr, K2CO3, DMF,
room temperature, 24 h, 97%; (e) MeNO2, KF, i-PrOH, NMM, room temperature, 48 h; (f) Ac2O, NaOAc, room temperature, 48 h, 72% (two steps); (g) Fe, SiO2, AcOH, toluene, 30 min,
reflux, 92%; (h) HNMe2, CH2O, AcOH, EtOH, 0 ꢁC/room temperature, 15 h, 97–99%; (i) (Ph)2C]NCH2CO2Et, n-BuP3, MeCN, reflux, 6 h, 82%; (j) HCl, H2O, THF, 4 h, room temperature
100%; (k) Boc2O, NaOH, H2O, THF, room temperature, 24 h, 100%; (l) LiOH, H2O, THF, 21 h, room temperature, 100% (three steps).
efficiently by a treatment with trimethyloxonium tetrafluoroborate
and Cs2CO3 in dichloromethane to give 47b. Next, the indole
nitrogen was protected as the corresponding N-tert-Boc derivative,
and then the silyl ether was removed with tetra-n-butylammonium
fluoride to provide 49b. Standard conditions for allylic chloride
formation provided 50b in good yield. The analogous reaction se-
quence was carried out for the syn-dioxopiperazine 46a.
The stage was now set for the critical intramolecular SN20 cy-
clization that sets the relative stereochemistry at C-20 during
formation of the bicyclo[2.2.2]diazaoctane ring nucleus. Based on
precedent from our paraherquamide A and B syntheses, anti-50b
was treated with NaH in refluxing benzene. However, no SN20
reaction took place and only epimerization of the tryptophan
residue was observed. We have previously observed in our
paraherquamide B synthesis that the SN20 reaction is highly tem-
perature dependent, and we speculated that higher temperatures
might allow the present substrate to reach a reactive conformation
for the SN20 reaction. Indeed, treatment of anti-50b with NaH in
refluxing xylene afforded the desired SN20 reaction product 51 in
77% yield exclusively as the desired syn-isomer (Scheme 5).
Compound syn-50a also underwent the same transformation to
give 51 in 56% yield. In practice, we found it convenient to employ
the anti/syn-diastereomeric mixture of 50, which was carried
through the subsequent steps leading to 51.
was the desired product, which was missing the O-benzyl residue.
Nonetheless, the large excess of NaBH4 resulted in a virtually
quantitative reduction of the s-palladium hexacyclic intermediate,
and as a result, after re-benzylation, 55 was obtained in an excellent
yield of 94% over two steps. The analogous transformation was
carried out using 10 equiv NaBD4 (99% atom D) in EtOD and pro-
vided the deuterium-labeled analogue 56 in 89% yield as a single
diastereomer (note that a new stereogenic center is created in this
process due to deuteration of the diastereotopic methyl group).
The incorporation of the deuterium in 56 was determined to be
93% atom D, most likely a result of the reaction of traces of the
s-palladium intermediate with EtOH used during the work-up and/
or Pd-mediated H/D exchange on the glass reaction vessel surface.
In the paraherquamide A synthesis, conditions could not be
found, which would allow direct and high-yielding conversion of
the analogous lactim ether 55 to the amide 59. However, 0.1 N
aqueous HCl in THF gave the corresponding ring-opened amine
methyl ester, which was cyclized with a catalytic amount of 2-
hydroxypyridine in hot toluene. Employing the same procedure
here, the conversion of the lactim ether 55 to the secondary
amide 59 was effected using 0.1 N aqueous HCl in THF to provide
in this case, a mixture of the secondary amide and the respective
amine methyl ester, which was directly cyclized to the bicy-
cle[2.2.2]diazaoctane by treatment with 2-hydroxypyridine in
refluxing toluene. The analogous transformation was carried out
with 56 to provide the deuterium-labeled analogue 60 in 86% over
the two steps.
Next, the chemoselective reduction of the tertiary amide in the
presence of the secondary amide to give 61 was investigated.
Treatment of 59 with 4–5 equiv DIBAL-H in toluene at room tem-
perature provided 61. Since the analogous deuterated reagent
DIBAL-D is neither commercially available nor reported as a solu-
tion in toluene, we prepared this important reagent based on the
procedure developed for the synthesis of dineopentylaluminum
hydride in toluene.36 Accordingly, triisobutylaluminum was
In our previously recorded paraherquamide
A synthesis,
Wacker-type cyclizations26c,d were utilized to form the sixth ring
using PdCl2, AgBF4, and propylene oxide in acetonitrile, however, in
the current case, these reaction conditions led to several by-prod-
ucts. Eventually, it was found, that the conversion of 52 to hexacycle
53 could be carried out successfully when Pd(CH3CN)4(BF4)2 was
utilized or preferably, Pd(TFA)2 in acetonitrile. As a result, closure of
the sixth ring was carried out using Pd(TFA)2 in acetonitrile/pro-
pylene oxide followed by NaBH4 (5 equiv) in EtOH to reduce the
incipient hexacyclic
s-palladium adduct (Scheme 6). Under these
conditions, the main product observed following the NaBH4 quench

3250
K. Sommer, R.M. Williams / Tetrahedron 65 (2009) 3246–3260
Me
CO2Et
OTBS
+
Me
TBSO
a
CO2H
Me
HN
Me
b
O
NHBoc
NHBoc
NH
EtO
32
N
OMe
OBn
N
BnO
44
O
H
OMe
45
Me
Me
TBSO
TBSO
OMe
N
O
Me
Me
NH
NH
c
d
NH
*
OMe
OBn
OMe
OBn
N
N
*
O
O
47a syn
47b anti
46a syn
46b anti
Me
Me
TBSO
HO
OMe
OMe
Me
Me
NBoc
NBoc
N
*
N
*
OMe
OMe
OBn
e
N
N
OBn
O
O
48a syn
48b anti
49a syn
49b anti
Me
OMe
Cl
Me
NBoc
N
*
OMe
OBn
f
N
O
50a syn
50b anti
Scheme 5. (a) pyBOP, DIEA, CH2Cl2, room temperature, 4 days, 84–89% (anti/syn-DKP); (b) TMSOTf, 2,6-lutidine, CH2Cl2, 1 h, room temperature, then NH4Cl, H2O, 50 min, room
temperature; then 2-HO-py, toluene, reflux, 12 days, 98% (anti-DKP), 67% (syn-DKP); (c) Me2OBF4, Cs2CO3, CH2Cl2, room temperature, 20 h, 99% (anti-DKP), 71% (syn-DKP); (d)
(Boc)2O, NEt3, DMAP, CH2Cl2, room temperature, 25 h, 92% (anti-DKP), 79% (syn-DKP); (e) n-Bu4NF, THF, room temperature, 24 h, 94% (anti-DKP), 92% (syn-DKP); (f) MsCl, LiCl, 2,4,6-
collidine, DMF, 0 ꢁC/room temperature, 5 days, 86% (anti-DKP), 86% (syn-DKP) or MsCl, 2,4,6-collidine, CH2Cl2, 0 ꢁC/room temperature, 24 h, then Bn(Bu)3NCl, DMF, 0 ꢁC/room
temperature, 22 h, 95% (anti-DKP).
reduced with LiAlD4 in heptanes under refluxing conditions and the
solvent replaced with toluene to provide the required reagent.
Freshly prepared toluene solutions of DIBAL-D effected the che-
moselective reduction of the tertiary amide in the presence of the
secondary amide furnishing hexacycle 62. After an initial reduction
with 4 equiv DIBAL-D yielded a mixture of the desired product and
the starting material, successive reductions of the crude material
with additional DIBAL-D gave the triply deuterated material in 52%
yield as a 5.7:1 inseparable mixture of Boc-protected product 62
and the corresponding product that had suffered loss of the N-tert-
Boc residue.
To reach the triply deuterium-labeled 7-hydroxy-pre-para-
herquamide, the benzyl protecting group of 62 was removed via
catalytic hydrogenation in the presence of Pd/C and the resulting
crude phenol was converted into the corresponding 6-mesylate 64
(Scheme 7). The reductive removal of the oxygen atom at the C6-
indole position proved troublesome. The C-6-mesylate 64
displayed only limited reactivity toward catalytic hydrogenation
and was reduced only when exposed to a large molar excess of
Pd(OH)2/C. Not surprisingly, these conditions resulted in the
concomitant reduction of the indole moiety to the corresponding
dihydroindole species 65. The Boc protecting group of 65 was
removed under standard reaction conditions to give 66. Cleavage
of the methyl ether was affected with BBr3 in CH2Cl2 and resulted
in a mixture of air sensitive compound 67 (major) and the
oxidation product, namely, the desired deuterium-labeled
7-hydroxy-pre-paraherquamide 27 (minor). Finally, when exposed
to air in a biphasic EtOAc/satd NaHCO3 solution, the mixture was
readily oxidized to give 27 exclusively.
The deuterated 7-hydroxy-pre-paraherquamide (27) possessed
the unfortunate property of being insoluble in water thus rendering
the planned feeding experiments challenging. To circumvent this
problem, the detergent TWEEN 80 was added and was found to
increase the miscibility of the substances in the culture broth
without inhibiting the production of paraherquamide A.
Feeding experiments were performed on P. fellutanum
(ATCC20841) followed by isolation and purification of
paraherquamide A. Within the limits of detection by 2H NMR
spectroscopy no incorporation was observed for the intact triply
labeled material 27. However, the isolated paraherquamide A
sample (14 mg) revealed the incorporation of one of the two
geminal deuterons of the CD2-group (2H NMR, 500 MHz, CHCl3/
CH3OH: 2.4 ppm; see Fig. 3). The lack of signals for the second
deuteron of the CD2-group (w3.6 ppm) and for the CH2D-group
(w1.2 ppm) suggests that 27 suffered catabolic degradation and
metabolic re-uptake of the deuteron. A rationalization for how
deuterium atoms from triply labeled II could be removed, and then
selectively re-incorporated at C–X of paraherquamide A is currently
lacking and more experimental data will need to be secured to
address this puzzling observation.
The implications of these observations are considerable. Since
the deuterated 7-hydroxy-pre-paraherquamide (27) was not

K. Sommer, R.M. Williams / Tetrahedron 65 (2009) 3246–3260
3251
Cl
the indole nucleus. The syntheses of the regioisomeric 6-hydroxy-
pre-paraherquamide (66) and the corresponding spiro-oxindole
(68) derived from pre-paraherquamide itself, as well as their cor-
responding N-methyl derivatives (67 and 69) are currently under
study to evaluate these alternatives.
OMe
R
N
Me
OMe
Me
OBn
c
Me
a
H
H
N
OMe
N
NBoc
Me
OMe
N
51: R=H
52: R=Boc
N
b
O
O
OBn
50a syn
50b anti
Boc OMe
N
Boc
N
OMe
OBn
OR2
e
Me
Me
RCH2
H
R1CH2
H
MeO2C
H
H
OMe
N
Me
Me
NH2
N
57: R = H
58: R = D
N
O
O
1
2
1
2
53: R1=H, R2=H or 54: R =D, R =H
d
1
2
55: R =H, R =Bn or 56: R =D, R =Bn
Boc OMe
Boc OMe
N
Me
N
OBn
g
RCH2
H
Me
R
f
OBn
RCH2
H
H
H
O
H
Me
O
H
N
N
Me
Figure 3. 2H NMR (500 MHz, CH3OHþCHCl3þ0.1%CDCl3/650
m
L) spectrum of the iso-
N
59 :R = H
60: R = D
61: R = H
62: R = D
N
O
lated sample of paraherquamide A from feeding experiment with P. fellutanum.
R
Scheme 6. (a) NaH, xylenes, 135 ꢁC, 60 min; (b) Boc2O, DMAP, NEt3, CH2Cl2, room
temperature, days, 77% (from the anti-diastereomer), 56% (from the syn-di-
astereomer); (c) Pd(OTFA)2, MeCN, propylene oxide, room temperature, 18–24 h, then
EtOH/NaBH4 or EtOD/NaBD4, room temperature, 3–8 h; (d) BnBr, K2CO3, DMF, room
temperature, 2–3 days, 63–94% (two steps); (e) 0.1 N HCl, THF, 0 ꢁC/room tempera-
ture, 80–90 min; (f) 2-HO-py, toluene, reflux, 3–5 h, 81–86% (two steps); (g) DIBAL-H
or DIBAL-D/toluene, ꢀ78 ꢁC/room temperature, 2–5 days, 52–77%.
The apparent transfer of the single deuterium atom from triply
labeled substrate 27 to paraherquamide A might be rationalized by
considering that this species serves as a non-endogenous substrate
for a reductase that employs NADH as a co-factor. Deuterium
transfer from 27 to the oxidized NADþ co-factor and subsequent
reduction of the endogenous substrate en route to paraherquamide
A might be one of many reasonable explanations for this un-
expected deuterium incorporation.
2
incorporated intact, this raises numerous provocative questions
concerning the timing and the regioselectivity of the hydroxylation
of the tryptophan-derived indole moiety for ultimate construction
of the dioxepin (for paraherquamide A and congeners) and the
pyran system (for paraherquamides F and G). The lack of in-
corporation of the intact triply labeled species 27 may indicate that
hydroxylation of the indole C-6 position (see Scheme 8, 66) likely
precedes hydroxylation of the indole C-7 position. It is also plau-
sible that oxidative processing of pre-paraherquamide to the
oxindole species 68 precedes hydroxylation at either C-6 or C-7 of
H
Me
N
Me
H
H
O
H
Me
N
N
20, pre-paraherquamide
[ox]
[ox]
?
H
O
Me
N
Me
OH
Me
H
Me
NH
H
H
X
Y
H
O
R
O
R
N
Me
Me
OMe
OMs
Boc
N
Boc
N
OMe
OR
N
2HCH2
H
Me
2H
Me
2HCH2
H
N
66: 6-hydroxy-pre-paraherquamide; R = H
N
68:R = H
69:R = Me
H
H
67: R = Me
O
O
Me
H
H
Me
b
N
N
2H
N
H
H
N
O
64
Me
2H
Me
Me
2H
62: R=Bn
63: R=OH
2H
a
NH
H
O
O
OH
Boc
OMe
O
N
Me
N
Me
OMe
OH
H
N
Me
2HCH2
H
N
Me
Me
2HCH2
H
c
d
f
H
1, paraherquamide A
O
H
Me
H
O
Me
Me
N
2H
Scheme 8. Some alternative biosynthetic transformations from pre-paraherquamide
(17) to paraherquamide A.
N
N
N
2H
65
66
2H
OH
H
H
Me
N
3. Conclusion
2HCH2
H
Me
N
2HCH2
H
e
H
In summary, we successfully prepared the putative biosynthetic
intermediate 7-hydroxy-pre-paraherquamide (27) in deuterium-
labeled form. This substance was used in a feeding experiment in
the paraherquamide-producing organism P. fellutanum. The iso-
lated sample of paraherquamide A indicated incorporation of only
one of the two geminal deuterons of the CD2-group (C-12). The lack
of signals for the second deuteron of the CD2-group at C-12 and for
the CH2D-group (C-22/C-23) suggests an unusual catabolic pro-
cessing of this substrate.
O
H
Me
O
H
N
2H
2H
N
2H
67
N
27
N
2H
Scheme 7. (a) H2, Pd/C (10%), EtOH, room temperature, 19 h; (b) MsCl, py, CH2Cl2, 0 ꢁ
C
to room temperature, 12–48 h, 64–89% (two steps); (c) H2, Pd(OH)2/C, MeOH, room
temperature, 3 days, 47%; (d) TFA, CH2Cl2, 0 ꢁC, 3 h, 98%; (e) (1) BBr3, CH2Cl2, ꢀ78 ꢁC to
room temperature, 13 h; (2) MeOH, ꢀ78 ꢁC to room temperature, 1 h; 3) NaHCO3, H2O,
room temperature, 30 min, >99% (BRSM), 87% conversion; (f) EtOAc, satd NaHCO3, air,
48 h, >99%.

3252
K. Sommer, R.M. Williams / Tetrahedron 65 (2009) 3246–3260
The synthetic technology developed herein, also provides ad-
TLC (SiO2, hexanes/EtOAc: 2:3): Rf¼0.13.
vanced synthetic intermediates that should be useful in the
asymmetric total synthesis of 6-hydroxy-pre-paraherquamide as
well as potential intermediates for the synthesis of para-
herquamides E and F. Efforts along these lines are currently in
progress in our laboratory and will be reported on in due course.
IR (thin film): 3269, 2957, 2844, 1669, 1578, 1508, 1353, 1321,
1268, 1193 cmꢀ1. 1H NMR (300 MHz, CDCl3)
d 3.99 (s, 3H), 6.45 (s,
1H), 7.22 (d, J¼8.4 Hz, 1H), 7.67 (d, J¼8.6 Hz, 1H), 9.81 (s, 1H). 13C
NMR (75 MHz, CD3OD): 188.2, 159.0, 141.3, 130.8, 123.2, 120.8, 62.6.
HRMS (FABþ) calcd for C8H7NO5 (m/z) 198.0402 [Mþþ1]; found
(m/z) 198.0399.
4. Experimental
4.4. 4-Benzyloxy-3-methoxy-2-nitro-benzaldehyde (37)
4.1. General methods
To a degassed solution of 36 (37.2 g, 189 mmol) in dry DMF
(350 mL) under argon were added anhydrous K2CO2 (39.1 g,
283 mmol) and benzyl bromide (38.7 g, 226 mmol). The reaction
mixture was stirred at room temperature for 24 h, poured into
water (4000 mL), the precipitate filtered off, and washed with
water (2000 mL). The resulting solid was dried under vacuum and
crystallized from ethyl acetate to give 4-benzyloxy-3-methoxy-2-
nitro-benzaldehyde (37) (52.4 g, 97%) as pale yellow crystals.
TLC (SiO2, hexanes/CH2Cl2: 1:4): Rf¼0.53.
Unless otherwise noted, materials were obtained from
commercial sources and utilized without purification. All reactions
requiring anhydrous conditions were performed under argon using
flame-dried glassware. Tetrahydrofuran, dimethylformamide, and
toluene were degassed with argon and passed through a solvent
purification system (J.C Meyer of Glass Contour) containing alu-
mina or molecular sieves. Dichloromethane was distilled from CaH2
prior to use. Column chromatography was performed on Merck
silica gel Kieselgel 60 (230–400 mesh). Mass spectra were obtained
on Fisons VG Autospec. HPLC data were obtained on a Waters 600
HPLC. 1H NMR, 13C NMR, and NOE experiments were recorded on
a Varian 300 or 400 MHz spectrometer. Chemical shifts were given
in parts per million and were recorded relative to the residual
solvent peak unless otherwise noted. 1H NMR signals were tabu-
lated in the following order: multiplicity (s, singlet; d, doublet; t,
triplet; q, quartet; and m, multiplet), coupling constant in hertz,
and number of protons. When a signal was deemed ‘broad’ it was
noted as such. IR spectra were recorded on a Nicolet Avatar 320 FT-
IR spectrometer. Optical rotations were determined with a Rudolph
Research Autopol III automatic polarimeter referenced to the D-line
of sodium.
IR (thin film): 3111, 3031, 2949, 2887, 1525, 1367, 1019, 953 cmꢀ1
.
1H NMR (300 MHz, CDCl3)
J¼8.6 Hz, 1H), 7.35–7.50 (m, 5H), 7.63 (d, J¼8.6 Hz, 1H), 9.80 (s, 1H).
d 3.99 (s, 3H), 5.27 (s, 2H), 7.18 (d,
13C NMR (75 MHz, CDCl3):
d 186.0, 157.7, 141.5, 135.0, 129.1 (2C),
128.9, 128.2 (2C), 127.6 (2C), 120.7, 114.5, 71.7, 62.5. HRMS (FABþ)
calcd for C15H13NO5 (m/z) 288.0872 [Mþþ1]; found (m/z) 288.0866.
4.5. 1-Benzyloxy-2-methoxy-3-nitro-4-(2-nitro-vinyl)-
benzene (38)
To a cooled (0 ꢁC) solution of 37 (40.0 g, 139 mmol) in a mixture
of DMF (100 mL), isopropanol (150 mL), and N-methylmorpholine
(140 mL) were added KF (4.05 g, 69.7 mmol) and nitromethane
(25.5 g, 418 mmol). The reaction mixture was stirred at room
temperature for 48 h. The solvent was removed in vacuo and the
resulting oil partitioned between water (2000 mL) and ethyl ether
(500 mL). The layers were separated and the aqueous layer
extracted with further ethyl ether (2ꢂ500 mL). The combined or-
ganic extracts were washed with brine and dried over anhydrous
Na2SO4. The solvent was removed in vacuo and the solid residue
was re-dissolved in acetic anhydride (190 mL). Sodium acetate
(4.00 g) was added and the resulting suspension stirred at room
temperature under argon for 2 days. The reaction mixture was
poured into ice (2000 g) and the resulting slurry was stirred for 2 h
until a fine suspension emerged. The precipitate was filtered off and
washed with water (2000 mL). The crude material was dried under
vacuum and crystallized from ethyl acetate/hexane to give 19.5 g of
the title compound. The remaining mother liquid was concentrated
in vacuo and purified by flash chromatography (silica, 6:4 hexane/
methylene dichloride then 5:5 hexane/methylene dichloride) to
yield further 13.6 g of 38 (33.1 g overall yield, 72%) as a pale yellow
solid.
4.2. Acetic acid 4-formyl-2-methoxy-phenyl ester (34)
To a stirred suspension of vanillin (40.0 g, 263 mmol) in dry
ethyl ether (100 mL) under argon was added slowly acetic anhy-
dride (39.1 mL, 408 mmol) and then DMAP (10 mg). The clarified
reaction mixture was stirred at room temperature for 20 h. The
crystallized white solid was filtered off and washed with water
(500 mL) to give 32.6 g of the title product. The aqueous mother
liquid was extracted with ethyl ether (3ꢂ200 mL), the combined
organic extracts were washed with water (3ꢂ250), and dried over
anhydrous MgSO4. The solvent was removed in vacuo to give fur-
ther 16.6 g of acetic acid 4-formyl-2-methoxy-phenyl ester 34
(49.2 g overall, 96%) as white solid.
TLC (SiO2, hexanes/EtOAc: 3:2): Rf¼0.52.
IR (thin film): 2966, 2846, 1750, 1686, 1277, 1204 cmꢀ1. 1H NMR
(CDCl3, 300 MHz)
d
2.34 (s, 3H), 3.90 (s, 3H), 7.22 (d, J¼7.9 Hz, 1H),
7.48 (dd, J¼9.3, 1.5 Hz, 2H), 9.94 (s, 1H). 13C NMR (75 MHz, CDCl3):
d
191.2, 168.5, 152.1, 145.0, 135.4, 124.8, 123.6, 111.0, 56.2, 20.8.
HRMS (FABþ) calcd for C10H10O4 (m/z) 195.0657 [Mþþ1]; found
(m/z) 195.0649.
TLC (SiO2, hexanes/CH2Cl2: 1:4): Rf¼0.33.
IR (thin film): 3111, 3031, 2949, 2887, 1525, 1367, 1019, 953 cmꢀ1
.
1H NMR (300 MHz, CDCl3)
d 4.00 (s, 3H), 5.25 (s, 2H), 7.12 (d,
4.3. 4-Hydroxy-3-methoxy-2-nitro-benzaldehyde (36)
J¼8.8 Hz, 1H), 7.33 (d, J¼8.8 Hz, 1H), 7.35–7.50 (m, 6H), 7.81 (d,
J¼13.6 Hz,1H). 13C NMR (75 MHz, CDCl3):
d 155.5,142.1, 138.5, 135.1,
To a stirred and cooled (ꢀ20 ꢁC) fuming nitric acid (240 mL) was
added slowly 34 (48.7 g, 251 mmol) over 1.5 h. The dark brown
reaction mixture was stirred for 3 h at ꢀ20 ꢁC and then poured into
2000 g of crushed ice. The yellow precipitate was filtered off and
washed with cold water (2000 mL). The resulting solid was dis-
solved in a solution of NaOH (15 g) in water (300 mL) and the dark
red solution stirred for 20 min. The solution was acidified (pH¼1)
with diluted hydrochloric acid (4 N, 250 mL), the precipitate fil-
tered off, washed with water, and dried in vacuo to give 36 (37.4 g,
76%) as yellow solid.
131.6, 129.1 (2C), 128.9 (2C), 127.5 (2C), 123.9, 115.6, 115.1, 71.7, 62.5.
HRMS (FABþ) calcd for C16H14N2O6 (m/z) 331.0930 [Mþþ1]; found
(m/z) 331.0942.
4.6. 6-Benzyloxy-7-methoxy-1H-indole (39)
1-Benzyloxy-2-methoxy-3-nitro-4-(2-nitro-vinyl)-benzene (38)
(10.8 g, 32.6 mmol), silica gel (70 g, 40–63 mm), and reduced iron
powder (31 g) were suspended in a degassed mixture of glacial
acetic acid (167 mL) and toluene (300 mL) under argon. The reaction

K. Sommer, R.M. Williams / Tetrahedron 65 (2009) 3246–3260
3253
mixture was heated under reflux for 45 min (pre-heated heating
cap) with efficient stirring. The fast cooled mixture (ice bath) was
filtered through a pad of silica and the solid material was washed
with ethyl ether (300 mL). Water (500 mL) was added and solid
NaHCO3 was carefully added in small portions until the aqueous
phase pH¼8.5. The organic phase was separated, the aqueous phase
was extracted with ethyl ether (300 mL), and the combined organic
phases were filtered through a pad of Na2SO4. The solvent was re-
moved in vacuo to yield 39 (7.79 g, 92%) as a yellow solid of sufficient
purity.
J¼8.5 Hz, 2H), 7.59 (d, J¼7.6 Hz, 2H), 8.00 (br s, 1H). 13C NMR
(75 MHz, CDCl3):
d 172.3, 146.0, 139.6, 138.0, 136.1, 135.4, 130.6,
130.4, 129.0, 128.7 (2C), 128.3 (2C), 128.2 (2C), 128.1 (2C), 128.0 (2C),
127.7 (2C), 127.7 (2C), 125.0, 122.9, 114.0, 112.6, 110.5, 73.0, 66.2,
61.2, 61.1, 29.5, 14.4. HRMS (FABþ) calcd for C34H32N2O4 (m/z)
533.2440 [Mþþ1]; found (m/z) 533.2431.
4.9. 2-Amino-3-(6-benzyloxy-7-methoxy-1H-indol-3-yl)-
propionic acid ethyl ester (42)
TLC (SiO2, hexanes/Et2O: 1:1): Rf¼0.40.
To a solution of 41 (1.00 g, 1.88 mmol) in THF (5 mL) was added
1 N HCl solution (18.8 mL, 18.8 mmol) and the reaction mixture was
stirred for 4 h at room temperature. THF was removed in vacuo and
the aqueous solution was extracted with diethyl ether (2ꢂ20 mL).
The aqueous phase was basified to pH¼8.5 with NaHCO3 and
extracted with methylene chloride (3ꢂ20 mL). Concentration un-
der reduced pressure gave 42 (699 mg, >99%) as an oil.
IR (thin film): 3428, 3063, 3031, 2934, 2864, 1504, 1443, 1378,
1293, 1228, 734 cmꢀ1. 1H NMR (300 MHz, CDCl3)
d 4.06 (s, 3H), 5.19
(s, 2H), 6.50 (dd, J¼3.3, 2.2 Hz, 1H), 6.90 (d, J¼8.4 Hz, 1H), 7.16 (dd,
J¼3.1, 2.2 Hz, 1H), 7.27 (dd, J¼8.4, 0.5 Hz, 1H), 7.30–7.45 (m, 3H),
7.51 (d, J¼7.3 Hz, 2H), 8.30 (br s, 1H). 13C NMR (75 MHz, CDCl3):
d
146.1, 137.9, 135.6, 130.6, 128.7 (2C), 128.0, 127.7 (2C), 125.0, 124.1,
115.6,111.2,102.9, 73.1, 61.2. HRMS (FABþ) calcd for C16H15NO2 (m/z)
254.1181 [Mþþ1]; found (m/z) 254.1177.
TLC (SiO2, CH2Cl2/MeOH: 9:1): Rf¼0.32.
IR (thin film): 3367, 3165, 3065, 2980, 2933, 2865, 1732, 1629,
1510, 1452, 1195, 1027 cmꢀ1 1H NMR (300 MHz, CDCl3)
. d 1.25 (t,
4.7. (6-Benzyloxy-7-methoxy-1H-indol-3-ylmethyl)-
dimethylamine (40)
J¼7.3 Hz, 3H), 1.83 (br s, 2H), 3.01 (1/2 ABX, J¼7.7 Hz, JAB¼14.3 Hz,
1H), 3.22 (1H, 1/2 ABX, J¼4.8 Hz, JAB¼14.3 Hz, 1H), 3.80 (dd,
J¼4.4 Hz, 7.3 Hz, 1H), 4.03 (s, 3H), 4.17 (q, J¼7.3 Hz, 2H), 5.17 (s, 2H),
6.89 (d, J¼8.4 Hz, 1H), 7.01 (d, J¼2.2 Hz, 1H), 7.23 (d, J¼8.4 Hz, 1H),
7.30–7.45 (m, 3H), 7.49 (d, J¼7.3 Hz, 2H), 8.24 (br s, 1H). 13C NMR
To a cooled (0 ꢁC) and stirred solution of 37% aqueous formal-
dehyde (4.47 mL, 0.060 mmol) and 40% aqueous dimethylamine
(7.54 mL, 0.060 mmol) in a mixture of glacial acetic acid (184 mL)
(75 MHz, CDCl3):
d 146.3, 137.9, 135.5, 131.1, 128.7 (2C), 128.0 (2C),
and ethanol (106 mL) was added
a
solution of 39 (6.08 g,
127.7 (2C), 124.8, 122.9, 113.8, 111.7, 110.7, 73.0, 61.1, 55.1, 31.0, 14.4.
HRMS (FABþ) calcd for C21H24N2O4 (m/z) 369.1814 [Mþþ1]; found
(m/z) 369.1816.
0.024 mmol) in ethanol (76 mL) and the reaction mixture stirred for
15 min at 0 ꢁC and then at room temperature for 15 h. The mixture
was diluted with water (300 mL) and made strongly basic with
a solution of NaOH (160 g) in water (1500 mL) while cooling in an
ice/water bath. The aqueous solution was extracted with ethyl ac-
etate (4ꢂ300 mL) and the combined organic extracts were dried
over anhydrous Na2SO4. The solvent was removed in vacuo and the
crude product was purified by flash chromatography (silica, 6:4
hexane/ethyl acetate then 4:6 hexane/ethyl acetate) to yield 40
(7.26 g, 97%) as a yellow solid.
4.10. 3-(6-Benzyloxy-7-methoxy-1H-indol-3-yl)-2-tert-
butoxycarbonylamino-propionic acid ethyl ester (43)
To a degassed solution of 42 (686 mg, 1.86 mmol) in dioxane
(10 mL) was added solid di-tert-butyl dicarbonate (448 mg,
2.05 mmol) followed by degassed aqueous 0.5 M NaOH solution
(3.73 mL, 1.86 mmol) and the reaction mixture was stirred for 14 h
at room temperature. The solvent was removed under reduced
pressure, the residue taken up in water (30 mL), and the suspension
acidified with 10% KHSO4 to pH 2. The mixture was extracted with
ethyl acetate (3ꢂ50 mL), the combined organic phases dried over
anhydrous Na2SO4, and the solvent removed under reduced pres-
sure to yield 43 (863 mg, >99%).
TLC (SiO2, hexanes/EtOAc: 1:1): Rf¼0.60.
IR (thin film): 3467, 3367, 3063, 3030, 2936, 2858, 1508, 1454,
1347, 1267, 1229, 1027 cmꢀ1 1H NMR (300 MHz, CDCl3)
. d 2.29 (s,
6H), 3.59 (s, 2H), 4.04 (3s, 3H), 5.17 (s, 2H), 6.90 (d, J¼8.8 Hz, 1H),
7.08 (d, J¼1.6 Hz, 1H), 7.20–7.45 (m, 4H), 7.49 (d, J¼7.3 Hz, 2H), 8.21
(br s, 1H). 13C NMR (75 MHz, CDCl3):
d 146.2, 137.9, 135.4, 130.9,
128.6 (2C), 127.9, 127.7 (2C), 125.2, 123.6, 114.3, 113.7, 110.7, 73.0,
61.1, 54.8, 45.5 (2C). HRMS (FABþ) calcd for C19H22N2O2 (m/z)
311.1760 [Mþþ1]; found (m/z) 311.1768.
TLC (SiO2, hexanes/EtOAc: 3:2): Rf¼0.34.
IR (thin film): 3359, 3304, 2971, 2929, 2829, 1735, 1691, 1520,
1229, 1159, 1024 cmꢀ1. 1H NMR (300 MHz, CDCl3) (rotamers)
d 1.21
(t, J¼7.3 Hz, 3H), 1.33, 1.43 (2s, 9H), 3.23 (d, J¼5.5 Hz, 2H), 4.03 (s,
3H), 4.13 (q, J¼7.3 Hz, 2H), 4.40, 4.61 (2dd, J¼5.6, 7.7 Hz, 1H), 5.09
(d, J¼8.1 Hz, 1H), 5.17 (s, 2H), 6.88 (d, J¼8.4 Hz, 1H), 6.95 (d,
J¼1.8 Hz, 1H), 7.17 (d, J¼8.4 Hz, 1H), 7.30–7.45 (m, 3H), 7.49 (d,
4.8. 2-(Benzhydrylidene-amino)-3-(6-benzyloxy-7-methoxy-
1H-indol-3-yl)-propionic acid ethyl ester (41)
To a suspension of 40 (100 mg, 0.322 mmol) and (benzhy-
drylidene-amino)-acetic acid ethyl ester (85.9 mg, 0.322 mmol)
in degassed and dry acetonitrile (2.5 mL) under argon was added
tri-n-butylphosphane (19.5 mg, 0.096 mmol), and the reaction
mixture was refluxed under argon for 6 h. After cooling to room
temperature, the solvent was removed in vacuo and the crude
product was purified by flash silica gel column chromatography
to yield 41 (141 mg, 82%) as an oil.
J¼7.7 Hz, 2H), 8.16 (br s, 1H). 13C NMR (75 MHz, CDCl3):
d 172.5,
155.4, 146.3, 137.9, 135.5, 130.9, 128.7 (2C), 128.0 (2C), 127.7 (2C),
125.1, 122.6, 113.9, 110.9, 79.9, 73.0, 61.5, 61.2, 54.4, 28.5 (3C), 28.3,
14.3. HRMS (FABþ) calcd for C26H32N2O6 (m/z) 468.2260; found
(m/z) 468.2243.
4.11. 3-(6-Benzyloxy-7-methoxy-1H-indol-3-yl)-2-tert-
butoxycarbonylamino-propionic acid (44)
TLC (SiO2, hexanes/EtOAc: 3:2): Rf¼0.41.
IR (thin film): 3368, 3060, 2977, 2932, 2864, 1733, 1623, 1576,
To a solution of 43 (852 mg, 1.82 mmol) in a degassed mixture
of THF and water (60 mL, 2:1) was added solid LiOH (218 mg,
9.10 mmol) at room temperature under argon. The reaction mix-
ture was stirred at room temperature for 6 h and the solvent
removed under reduced pressure. The resulting slurry was taken
in water (50 mL), acidified with 10% KHSO4 to pH 2, and extracted
with methylene dichloride (3ꢂ50 mL) and ethyl acetate
1508,1228,1027 cmꢀ1. 1H NMR (300 MHz, CDCl3)
d
1.24 (t, J¼7.0 Hz,
3H), 3.20 (1/2 ABX, J¼8.8 Hz, JAB¼14.3 Hz, 1H), 3.40 (1/2 ABX,
J¼4.4 Hz, JAB¼13.9 Hz, 1H), 4.01 (s, 3H), 4.10–4.25 (m, 2H), 4.34 (dd,
J¼4.8, 8.8 Hz, 1H), 5.15 (s, 2H), 6.61 (d, J¼6.6 Hz, 2H), 6.71 (d,
J¼8.8 Hz, 1H), 6.85 (d, J¼8.4 Hz, 1H), 6.90 (d, J¼2.2 Hz, 1H), 7.13 (t,
J¼7.7 Hz, 2H), 7.22 (d, J¼7.5 Hz, 1H), 7.30–7.45 (m, 6H), 7.49 (d,

3254
K. Sommer, R.M. Williams / Tetrahedron 65 (2009) 3246–3260
(1ꢂ50 mL). The combined organic phases were dried over anhy-
drous Na2SO4 and the solvent removed under reduced pressure to
afford 44 (805 mg, >99%).
Compound 46a: TLC (SiO2, CH2Cl2/MeOH: 9:1): Rf¼0.48.
25
[
a
]
D
ꢀ77.0 (c 0.40, CHCl3). IR (thin film): 3275, 3064, 2957, 2929,
2855, 1653, 1436, 1323, 1253, 1068, 1029, 837 cmꢀ1
.
1H NMR
TLC (SiO2, CH2Cl2/MeOH: 9:1): Rf¼0.20.
(300 MHz, CDCl3) 0.03 (s, 6H), 0.88 (s, 9H), 0.82–0.88 (m, 3H), 1.66
d
IR (thin film): 3341, 2977, 2933, 1700, 1509, 1256, 1229, 1067,
(s, 3H),1.55–1.70 (m, 2H), 2.15–2.40 (m, 2H), 2.40–2.55 (m, 1H), 2.74
(1/2 ADX, J¼8.9 Hz, JAB¼14.6 Hz, 1H), 2.95 (1/2 ABX, J¼11.5 Hz,
JAB¼14.9 Hz, 1H), 3.45 (1/2 ABX, J¼2.9 Hz, JAB¼12.5 Hz, 1H), 3.56 (1/
2 ABX, J¼3.3 Hz, JAB¼14.6 Hz, 1H), 4.03 (s, 3H), 4.09 (s, 2H), 4.19 (dt,
J¼3.2, 11.3 Hz, 1H), 5.17 (s, 2H), 5.52 (t, J¼8.2 Hz, 1H), 5.75 (d,
J¼2.1 Hz, 1H), 6.90 (d, J¼8.9 Hz, 1H), 7.01 (d, J¼2.1 Hz, 1H), 7.24 (d,
J¼8.4 Hz, 1H), 7.30–7.45 (m, 3H), 7.49 (d, J¼7.6 Hz, 2H), 8.23 (br s,
1027 cmꢀ1 1H NMR (300 MHz, CDCl3) (rotamers)
. d 1.31, 1.45 (2s,
9H), 3.08, 3.29 (2s, 2H), 4.01 (s, 3H), 4.47, 4.71 (2s, 1H), 5.16 (s, 2H),
6.85 (s, 1H), 6.88 (d, J¼8.8 Hz, 1H), 7.23 (d, J¼8.8 Hz, 1H), 7.30–7.45
(m, 3H), 7.48 (d, J¼7.3 Hz, 2H), 8.58 (br s, 1H). 13C NMR (75 MHz,
CDCl3):
d 176.5, 157.9, 147.2, 139.5, 137.0, 132.2, 129.6 (2C), 128.9,
128.9, 126.8, 124.5, 114.9, 111.9, 111.4, 80.6, 74.2, 61.5, 56.2, 29.0 (2C),
28.8, 28.5. HRMS (FABþ) calcd for C24H28N2O6 (m/z) 440.1947;
found (m/z) 440.1948.
1H). 13C NMR (75 MHz, CDCl3):
d 168.3, 165.4, 146.4, 139.7, 137.6,
135.5, 131.3, 128.6, 127.9, 127.6, 123.8, 123.4, 117.2, 113.8, 110.9, 110.7,
72.9, 72.4, 68.3, 61.2, 57.3, 43.2, 41.0, 35.6, 32.6, 27.5, 26.2, 26.2, 18.7,
16.9, 14.0, ꢀ4.98, ꢀ5.07. HRMS (FABþ) calcd for C36H49N3O5Si (m/z)
631.3442; found (m/z) 631.3442.
4.12. 1-[3-(6-Benzyloxy-7-methoxy-1H-indol-3-yl)-2-tert-
butoxycarbonylamino-propionyl]-2-[4-(tert-butyl-dimethyl-
silanyloxy)-3-methyl-but-2-enyl]-3-methyl-pyrrolidine-2-
carboxylic acid ethyl ester (45 syn/anti)
Compound 46b: TLC (SiO2, CH2Cl2/MeOH: 9:1): Rf¼0.27.
25
[
a
]
D
þ64.6 (c 0.57, CHCl3). IR (thin film): 3272, 3064, 2955,
2929, 2855, 1676, 1655, 1446, 1256, 1069, 1029, 838 cmꢀ1. 1H NMR
(300 MHz, CDCl3) 0.02, 0.04 (2s, 6H), 0.87 (s, 9H), 0.82–0.88 (m,
To a solution of BOP (402 mg, 951
mmol), 44 (419 mg, 951
mmol),
d
and 32 (307 mg, 864 mol) in dry CH2Cl2 (1.5 mL) was added DIEA
m
3H), 1.55 (s, 3H), 1.56–1.68 (m, 2H), 2.18–2.34 (m, 1H), 2.36–2.48 (m,
2H), 2.63 (1/2 ADX, J¼7.7 Hz, JAB¼13.9 Hz, 1H), 2.88 (1/2 ABX,
J¼10.2 Hz, JAB¼14.7 Hz,1H), 3.52 (1/2 ABX, JAB¼11.5 Hz,1H), 3.61 (1/
2 ABX, J¼3.3 Hz, JAB¼14.7 Hz, 1H), 3.78–3.88 (m, 1H), 3.93 (s, 2H),
4.03 (s, 3H), 4.28 (dd, J¼3.3, 9.9 Hz, 1H), 5.17 (s, 2H), 5.40 (t,
J¼7.7 Hz, 1H), 5.73 (s, 1H), 6.88 (d, J¼8.4 Hz, 1H), 7.01 (s, 1H), 7.19 (d,
J¼8.4 Hz, 1H), 7.30–7.44 (m, 3H), 7.46 (d, J¼7.7 Hz, 2H), 8.21 (br s,
(223 mg, 1.73 mmol) at room temperature and the reaction mixture
was stirred for 44 h. The solvent was removed in vacuo and the
residue was purified by flash column chromatography (silica, hex-
ane/ethyl acetate: 8:2 then 7:3 then 6:4 then 5:5 then 4:6 then 3:7
then 2:8 then 0:10) to yield 45 as a mixture of diastereomers
(474 mg, 71%) as an oil.
TLC (SiO2, hexanes/EtOAc: 3:2): Rf¼0.32.
1H). 13C NMR (75 MHz, CDCl3):
d 169.8, 165.3, 146.3, 140.1, 137.6,
IR (thin film): 3330, 2955, 2931, 2852, 1726, 1711, 1643, 1445,
1365, 1250, 1170, 1068, 837 cmꢀ1. 1H NMR (300 MHz, CDCl3) (mix-
135.4, 131.3, 128.6 (2C), 127.9, 127.6 (2C), 124.1, 123.5, 116.3, 113.8,
110.8, 110.3, 72.9, 72.7, 68.0, 61.1, 55.1, 43.1, 40.4, 36.4, 29.2, 27.8,
26.1 (3C), 18.6, 16.3, 13.8, ꢀ5.03, ꢀ5.07. HRMS (FABþ) calcd for
C36H49N3O5Si (m/z) 631.3442; found (m/z) 631.3437.
ture of two diastereomers and two rotamers) d 0.04 (s, 6H), 0.89 (s,
9H), 0.89–0.98 (m, 3H), 1.20–1.32 (m, 3H), 1.37, 1.39 (2s, 9H), 1.62 (s,
3H),1.50–1.65 (m,1H),1.68–1.82 (m,1H), 2.10–2.30 (m,1H), 2.65 (dd,
J¼8.0, 15.2 Hz, 1H), 2.86–3.46 (m, 5H), 3.85–4.00 (m, 2H), 4.02 (s,
3H), 4.06–4.28 (m, 2H), 4.62–4.74, 4.82–4.92 (2 m, 1H), 5.17 (s, 2H),
5.24 (t, J¼9.2 Hz, 1H), 6.88 (t, J¼8.4 Hz, 1H), 6.95, 7.14 (2s, 1H), 7.24–
7.42 (m, 4H), 7.49 (d, J¼7.0 Hz, 2H), 8.14, 8.37 (2br s, 1H). 13C NMR
(75 MHz, CDCl3): (mixture of two diastereomers and two rotamers)
4.14. syn-3S-(6-Benzyloxy-7-methoxy-1H-indol-3-ylmethyl)-
8aR-[4-(tert-butyl-dimethyl-silanyloxy)-3-methyl-but-2E-
enyl]-1-methoxy-8S-methyl-6,7,8,8a-tetrahydro-3H-
pyrrolo[1,2-a]pyrazin-4-one (47a and 47b)
d
171.9,170.8,170.4,155.3,155.2,146.1,146.0,138.7,138.7,137.8,137.8,
To a solution of 46a and 46b (2.36 g, 3.73 mmol) in dry CH2Cl2
(100 mL) was added Cs2CO3 (24.3 g, 74.7 mmol) and the reaction
mixture was stirred for 30 min at room temperature. Me3OBF4
(2.76 g, 18.7 mmol) was added in one portion and the stirring
continued for 20 h. The reaction mixture was poured into satd
NaHCO3 solution (150 mL), the organic phase was separated, and
the aqueous phase extracted with ethyl acetate (3ꢂ50 mL). The
combined organic phases were dried over Na2SO4, the solvent was
removed in vacuo, and the residue was purified by flash column
chromatography (silica, hexane/ethyl acetate: 8:2 then 7:3) to yield
47a (1.16 g, 48%) and 47b (560 mg, 23%) both as a foam.
135.4,135.4,130.7,130.7, 128.6, 128.6, 127.9,127.9, 127.6, 127.6, 125.1,
125.0, 123.1, 123.1, 122.9, 122.9, 119.7, 119.6, 118.9, 118.9, 113.8, 111.2,
111.1, 110.7, 110.5, 79.5, 79.5, 73.0, 72.9, 72.2, 72.0, 69.0, 61.2, 61.0,
60.9, 52.6, 52.5, 48.4, 47.8, 40.1, 39.8, 31.5, 31.4, 29.6, 29.5, 8.8, 28.6,
26.2, 26.2, 18.7,14.7, 14.6,14.4,14.2, 14.2, ꢀ4.85, ꢀ4.91. HRMS (FABþ)
calcd for C43H63N3O8Si (m/z) 777.4385; found (m/z) 777.4399.
4.13. 3-(6-Benzyloxy-7-methoxy-1H-indol-3-ylmethyl)-8a-[4-
(tert-butyl-dimethyl-silanyloxy)-3-methyl-but-2-enyl]-8-
methyl-hexahydro-pyrrolo[1,2-a]pyrazine-1,4-dione
(46a and 46b)
TLC (SiO2, hexanes/EtOAc: 1:4): Rf¼0.30 (syn).
TLC (SiO2, hexanes/EtOAc: 1:4): Rf¼0.55 (anti).
25
To a solution of 45 (457 mg, 588
mmol) in dry CH2Cl2 (7 mL) was
Compound 47a: [
a
]
D
þ8.33 (c 0.42, CHCl3). IR (thin film): 3287,
added 2,6-lutidine (173 mg, 1.62 mmol) followed by TMSOTf
(261 mg, 1.18 mmol) and the reaction mixture was stirred for 1 h at
room temperature. The reaction mixture was quenched with satd
aqueous solution of NH4Cl (10 mL), stirred for 45 min, and 10%
aqueous NaHCO3 solution (20 mL) was added. The organic phase
was separated and the aqueous phase extracted with ethyl acetate
(3ꢂ50 mL). The combined organic phases were dried over anhy-
drous Na2SO4, the solvent was removed in vacuo, and the residue
was dissolved in dry toluene and 2-hydroxypyridine (53.5 mg,
2947, 2929, 2855, 1692, 1645, 1447, 1253, 1069, 837 cmꢀ1. 1H NMR
(300 MHz, CDCl3)
0.00 (s, 6H), 0.73 (d, J¼6.8 Hz, 3H), 0.87 (s, 9H),
d
1.45–1.60 (m, 1H), 1.51 (s, 3H), 1.82 (dd, J¼7.1, 16.0 Hz, 1H), 2.00–
2.20 (m, 2H), 2.20–2.40 (m, 1H), 2.98 (dd, J¼8.0, 14.6 Hz, 1H), 3.27
(1/2 ABX, JAB¼11.9 Hz, 1H), 3.40 (1/2 ABX, J¼4.2 Hz, JAB¼14.1 Hz,
1H), 3.65 (s, 3H), 3.80–3.20 (m, 3H), 3.98 (s, 3H), 4.38 (dd, J¼3.0,
8.4 Hz, 1H), 5.15 (s, 2H), 5.29 (t, J¼7.8 Hz, 1H), 6.83 (d, J¼8.2 Hz, 1H),
7.06 (d, J¼1.5 Hz, 1H), 7.30–7.45 (m, 4H), 7.47 (d, J¼8.1 Hz, 2H), 8.20
(br s, 1H). 13C NMR (75 MHz, CDCl3):
d 169.4, 159.7, 145.9, 138.2,
562
m
mol) was added. The reaction mixture was heated under
137.9, 135.4, 130.6, 128.5 (2C), 127.8, 127.6 (2C), 125.4, 122.7, 118.0,
114.7, 113.7, 110.6, 73.1, 70.3, 68.2, 62.8, 61.1, 52.8, 42.0, 39.6, 35.0,
32.0, 27.8, 26.2 (3C), 18.6, 16.9, 13.7, ꢀ5.06, ꢀ5.09. HRMS (FABþ)
calcd for C37H51N3O5Si (m/z) 646.3654 [Mþþ1]; found (m/z)
646.3654.
reflux for 12 days. The solvent was removed in vacuo and the res-
idue was purified by flash column chromatography (silica, CH2Cl2/
MeOH: 100:0 then 98:2 then 96:4) to yield 46b (116 mg, 49%) as an
oil and 46a (78 mg, 34%) as an oil.

K. Sommer, R.M. Williams / Tetrahedron 65 (2009) 3246–3260
3255
25
Compound 47b: [
2953, 2929, 2855, 1698, 1635, 1452, 1252, 1068, 837 cmꢀ1. 1H NMR
(300 MHz, CDCl3)
a
]
þ97.6 (c 0.52, CHCl3). IR (thin film): 3291,
TLC (SiO2, hexanes/EtOAc: 2:3): Rf¼0.65.
25
D
[a
]
þ5.95 (c 0.42, CHCl3). IR (thin film): 2933, 2856,1753, 1695,
D
d
ꢀ0.12 (d, J¼7.0 Hz, 3H), 0.05 (s, 6H), 0.90 (s, 9H),
1652, 1436, 1352, 1235, 1157, 1043, 837 cmꢀ1 1H NMR (300 MHz,
.
1.25–1.35 (m, 1H), 1.53 (s, 3H), 2.00–2.15 (m, 2H), 2.23 (1/2 ADX,
J¼8.5 Hz, JAB¼14.9 Hz,1H), 2.33 (1/2 ABX, J¼8.2 Hz, JAB¼14.9 Hz,1H),
3.20–3.40 (m, 2H), 3.49 (1/2 ABX, J¼5.1 Hz, JAB¼14.7 Hz,1H), 3.62 (s,
3H), 3.55–3.70 (m,1H), 3.95 (s, 2H), 3.96 (s, 3H), 4.26 (t, J¼4.4 Hz,1H),
5.15 (s, 2H), 5.21 (dt, J¼1.5, 8.2 Hz,1H), 6.80 (d, J¼8.2 Hz,1H), 6.97 (d,
J¼2.1 Hz,1H), 7.30–7.45 (m, 4H), 7.46 (d, J¼7.5 Hz, 2H), 7.98 (br s,1H).
CDCl3)
d
0.00 (s, 6H), 0.09 (d, J¼6.3 Hz, 3H), 0.86 (s, 9H), 1.25–1.40
(m, 1H), 1.49 (s, 3H), 1.59 (s, 9H), 2.00–2.17 (m, 2H), 2.22 (1/2 ADX,
J¼8.4 Hz, JAB¼14.2 Hz, 1H), 2.33 (1/2 ABX, J¼8.3 Hz, JAB¼14.6 Hz,
1H), 3.22 (1/2 ABX, J¼4.1 Hz, JAB¼14.7 Hz, 1H), 3.33 (1/2 ABX,
J¼5.1 Hz, JAB¼14.7 Hz, 1H), 3.57 (s, 3H), 3.55–3.70 (m, 1H), 3.85 (s,
3H), 3.90 (s, 2H), 4.21 (t, J¼4.4 Hz, 1H), 5.13 (s, 2H), 5.19 (t, J¼8.2 Hz,
1H), 6.90 (d, J¼8.8 Hz, 1H), 7.20–7.40 (m, 4H), 7.44 (d, J¼7.6 Hz, 2H).
13C NMR (75 MHz, CDCl3):
d 168.6, 159.1, 145.5, 139.3, 137.9, 135.1,
130.2, 128.5 (2C), 127.8, 127.7 (2C), 126.1, 123.2, 117.0, 115.6, 113.0,
110.2, 73.1, 70.5, 68.2, 61.3, 61.1, 52.6, 41.6, 39.7, 35.3, 29.3, 28.3, 26.2
(3C),18.6,15.0,13.7, ꢀ5.05 (2C). HRMS (FABþ) calcd for C37H51N3O5Si
(m/z) 645.3598; found (m/z) 645.3601.
13C NMR (75 MHz, CDCl3):
d 168.5, 159.5, 149.6, 148.8, 139.4, 139.1,
137.9, 129.7, 128.7 (2C), 128.5, 127.7, 127.7 (2C), 126.7, 116.7, 116.7,
115.5, 113.5, 82.7, 73.3, 70.7, 68.1, 61.3, 60.5, 52.6, 41.7, 39.7, 35.3,
28.5, 28.4 (3C), 26.2 (3C), 18.6, 15.6, 13.7, ꢀ5.07 (2C). HRMS (FABþ)
calcd for C42H59N3O7Si (m/z) 746.4201 [Mþþ1]; found (m/z)
746.4202.
4.15. syn-6-Benzyloxy-3-{8aR-[4-(tert-butyl-dimethyl-
silanyloxy)-3-methyl-but-2E-enyl]-1-methoxy-8S-methyl-
4-oxo-3,4,6,7,8,8a-hexahydro-pyrrolo[1,2-a]pyrazin-3S-
ylmethyl}-7-methoxy-indole-1-carboxylic acid
tert-butyl ester (48a)
4.17. syn-6-Benzyloxy-3-[8aR-(4-hydroxy-3-methyl-but-2E-
enyl)-1-methoxy-8S-methyl-4-oxo-3,4,6,7,8,8a-hexahydro-
pyrrolo[1,2-a]pyrazin-3S-ylmethyl]-7-methoxy-indole-1-
carboxylic acid tert-butyl ester (49a)
To a solution of 47a (1.09 g, 1.69 mmol) in dry CH2Cl2 (15 mL)
was added triethyl amine (188 mg, 1.86 mmol) followed by DMAP
(206 mg, 1.69 mmol) and the reaction mixture was stirred for
To a solution of 48a (980 mg, 1.31 mmol) in dry THF (13 mL) was
added a 1 M solution of TBAF (4.33 mL, 4.33 mmol) in THF and the
reaction mixture was stirred for 24 h at room temperature. Most of
the solvent was removed in vacuo and satd NaHCO3 solution
(50 mL) was added, and then the reaction mixture was extracted
with ethyl acetate (3ꢂ30 mL). The combined organic phases were
dried over Na2SO4, the solvent was removed in vacuo, and the
residue was purified by flash column chromatography (silica,
methylene dichloride/methanol: 10:0 then 98:2 then 96:4) to yield
49a (780 mg, 92%) as a foam.
10 min at room temperature.
A solution of Boc2O (1.10 g,
5.06 mmol) in dry CH2Cl2 (5 mL) was added and the stirring con-
tinued for 72 h. The reaction mixture was poured into satd NaHCO3
solution (100 mL), the organic phase was separated, and the
aqueous phase extracted with ethyl acetate (3ꢂ50 mL). The
combined organic phases were dried over Na2SO4, the solvent was
removed in vacuo, and the residue was purified by flash column
chromatography (silica, hexane/ethyl acetate: 8:2 then 7:3 then 6:4
then 5:5 then 4:6) to yield 48a (990 mg, 79%) as a foam.
TLC (SiO2, CH2Cl2/MeOH: 95:5): Rf¼0.35.
25
TLC (SiO2, hexanes/EtOAc: 2:3): Rf¼0.30.
[a
]
ꢀ11.3 (c 0.39, CHCl3). IR (thin film): 3406, 2974, 2942, 1751,
D
25
[
a]
ꢀ17.8 (c 0.46, CHCl3). IR (thin film): 2950, 2931, 2855, 1753,
1695, 1635, 1456, 1354, 1261, 1157, 1026 cmꢀ1
.
1H NMR (300 MHz,
0.74 (d, J¼6.7 Hz, 3H), 1.59 (s, 3H), 1.63 (s, 9H), 1.50–1.70
D
1695, 1652, 1436, 1354, 1257, 1157, 1043, 837 cmꢀ1
(300 MHz, CDCl3)
.
1H NMR
ꢀ0.04, ꢀ0.03 (2s, 6H), 0.76 (d, J¼6.4 Hz, 3H),
CDCl3)
d
d
(m, 2H), 1.75–1.85 (br s, 1H), 2.00–2.20 (m, 2H), 2.25–2.40 (m, 2H),
2.82 (dd, J¼9.9, 15.1 Hz, 1H), 3.27 (1/2 ADX, J¼3.0 Hz, JAB¼13.9 Hz),
3.35 (1/2 ADX, J¼3.9 Hz, JAB¼15.6 Hz), 3.64 (s, 3H), 3.91 (s, 3H), 3.95
(s, 2H), 3.90–4.10 (m, 1H), 4.34 (dd, J¼3.9, 8.9 Hz, 1H), 5.16 (s, 2H),
5.22 (t, J¼7.8 Hz, 1H), 6.95 (d, J¼9.2 Hz, 1H), 7.22 (d, J¼8.9 Hz, 1H),
7.30–7.45 (m, 3H), 7.47 (d, J¼8.1 Hz, 2H). 13C NMR (75 MHz, CDCl3):
0.84 (s, 9H), 1.56 (s, 3H), 1.64 (s, 9H), 2.05–2.25 (m, 2H), 2.30–2.45
(m, 2H), 2.75 (dd, J¼10.1, 14.6 Hz, 1H), 3.25–3.45 (m, 2H), 3.64 (s,
3H), 3.92 (s, 3H), 3.98 (s, 2H), 3.90–4.20 (m, 1H), 4.31 (dd, J¼3.0,
8.9 Hz, 1H), 5.17 (s, 2H), 5.36 (t, J¼7.8 Hz, 1H), 6.94 (d, J¼8.2 Hz, 1H),
7.19 (d, J¼8.9 Hz, 1H), 7.30–7.45 (m, 3H), 7.48 (d, J¼8.1 Hz, 2H). 13C
NMR (75 MHz, CDCl3):
d
169.1,159.5,150.0,148.8,139.5,138.8,137.8,
d 169.1, 159.6, 150.0, 148.9, 139.4, 138.8, 137.8, 129.1, 129.0, 128.5
129.2, 129.0, 128.5 (2C), 127.8, 127.6 (2C), 126.3, 117.5, 117.4, 114.7,
113.8, 82.8, 73.4, 70.6, 68.0, 62.1, 61.5, 52.8, 42.0, 40.1, 34.8, 31.7, 28.3
(3C), 27.9, 26.1 (3C), 18.6, 17.0, 13.7, ꢀ5.13, ꢀ5.18. HRMS (FABþ) calcd
for C42H59N3O7Si (m/z) 746.4201 [Mþþ1]; found (m/z) 746.4187.
(2C), 127.8, 127.6 (2C), 126.4, 118.7, 117.2, 114.6, 113.7, 83.0, 73.3, 70.5,
68.1, 62.0, 61.4, 52.8, 42.2, 40.2, 35.1, 31.5, 28.3 (3C), 27.9, 17.0, 13.9.
HRMS (FABþ) calcd for C36H45N3O7 (m/z) 632.3336 [Mþþ1]; found
(m/z) 632.3304.
4.16. anti-6-Benzyloxy-3-{8aR-[4-(tert-butyl-dimethyl-
silanyloxy)-3-methyl-but-2E-enyl]-1-methoxy-8S-methyl-
4-oxo-3,4,6,7,8,8a-hexahydro-pyrrolo[1,2-a]pyrazin-3R-
ylmethyl}-7-methoxy-indole-1-carboxylic acid
tert-butyl ester (48b)
4.18. anti-6-Benzyloxy-3-[8aR-(4-hydroxy-3-methyl-but-2E-
enyl)-1-methoxy-8S-methyl-4-oxo-3,4,6,7,8,8a-hexahydro-
pyrrolo[1,2-a]pyrazin-3R-ylmethyl]-7-methoxy-indole-1-
carboxylic acid tert-butyl ester (49b)
To a solution of 48b (1.86 g, 2.49 mmol) in dry THF (25 mL) was
added a 1 M solution of TBAF (8.21 mL, 8.21 mmol) in THF and the
reaction mixture was stirred for 24 h at room temperature. The
most of solvent was removed in vacuo and satd NaHCO3 solution
(100 mL) was added, and then the reaction mixture was extracted
with ethyl acetate (3ꢂ50 mL). The combined organic phases were
dried over Na2SO4, the solvent was removed in vacuo, and the
residue was purified by flash column chromatography (silica,
methylene dichloride/methanol: 10:0 then 98:2 then 96:4) to yield
49b (1.47 g, 94%) as a foam.
To a solution of 47b (1.54 g, 2.38 mmol) in dry CH2Cl2 (20 mL)
was added triethyl amine (265 mg, 2.62 mmol) followed by DMAP
(291 mg, 2.38 mmol) and the reaction mixture was stirred for
10 min at room temperature.
A solution of Boc2O (1.56 g,
7.15 mmol) in dry CH2Cl2 (5 mL) was added and stirring continued
for 25 h. The reaction mixture was poured into satd NaHCO3 solu-
tion (100 mL), the organic phase was separated, and the aqueous
phase extracted with ethyl acetate (3ꢂ50 mL). The combined or-
ganic phases were dried over Na2SO4, the solvent was removed in
vacuo, and the residue was purified by flash column chromatog-
raphy (silica, hexane/ethyl acetate: 9:1 then 8:2 then 7:3 then 6:4)
to yield 48b (1.64 g, 92%) as a foam.
TLC (SiO2, hexanes/EtOAc: 2:3): Rf¼0.68.
25
[a
]
þ66.5 (c 0.32, CHCl3). IR (thin film): 3407, 2975, 2941, 2887,
D
1752, 1695, 1646, 1436, 1352, 1266, 1156, 1025 cmꢀ1 1H NMR
.

3256
K. Sommer, R.M. Williams / Tetrahedron 65 (2009) 3246–3260
(300 MHz, CDCl3)
d
0.07 (d, J¼6.3 Hz, 3H), 1.25–1.45 (m, 1H), 0.86 (s,
J¼7.9 Hz, JAB¼14.2 Hz, 1H), 2.36 (1/2 ABX, J¼8.3 Hz, JAB¼14.6 Hz,
1H), 3.24 (1/2 ABX, J¼4.1 Hz, JAB¼14.7 Hz, 1H), 3.36 (1/2 ABX,
J¼5.1 Hz, JAB¼14.7 Hz, 1H), 3.20–3.40 (m, 1H), 3.57 (s, 3H), 3.55–
3.70 (m, 1H), 3.86 (s, 3H), 3.91 (s, 2H), 4.30 (t, J¼4.6 Hz, 1H), 5.13 (s,
2H), 5.30 (t, J¼7.4 Hz,1H), 6.90 (d, J¼8.84 Hz,1H), 7.20–7.40 (m, 4H),
9H), 1.59 (s, 3H), 1.62 (s, 9H), 1.92 (br s, 1H), 2.00–2.20 (m, 2H), 2.24
(1/2 ADX, J¼7.9 Hz, JAB¼14.2 Hz, 1H), 2.37 (1/2 ABX, J¼8.3 Hz,
JAB¼14.6 Hz, 1H), 3.24 (1/2 ABX, J¼4.1 Hz, JAB¼14.7 Hz, 1H), 3.38 (1/
2 ABX, J¼5.1 Hz, JAB¼14.7 Hz, 1H), 3.20–3.40 (m, 1H), 3.59 (s, 3H),
3.55–3.70 (m, 1H), 3.87 (s, 3H), 3.95 (s, 2H), 4.28 (t, J¼5.1 Hz, 1H),
5.16 (s, 2H), 5.22 (t, J¼8.4 Hz, 1H), 6.92 (d, J¼8.8 Hz, 1H), 7.20–7.40
7.44 (d, J¼7.7 Hz, 2H). 13C NMR (75 MHz, CDCl3):
d 168.3, 159.1,
149.6, 148.8, 139.1, 137.8, 136.6, 129.6, 128.6 (2C), 128.4, 127.7, 127.6
(2C), 126.7, 123.2, 116.5, 115.4, 113.4, 82.7, 73.2, 70.4, 61.3, 60.5, 52.7,
51.6, 41.8, 39.8, 35.8, 28.6, 28.3 (3C), 15.4, 14.4. HRMS (FABþ) calcd
for C36H44ClN3O6 (m/z) 649.2919; found (m/z) 649.2933.
(m, 4H), 7.46 (d, J¼7.7 Hz, 2H). 13C NMR (75 MHz, CDCl3):
d 168.5,
159.5, 149.6, 148.8, 140.1, 139.1, 137.8, 129.6, 128.6 (2C), 128.5, 127.8,
127.6 (2C), 126.7, 117.7, 116.5, 115.4, 113.4, 82.8, 73.2, 70.6, 68.2, 61.3,
60.6, 52.7, 41.7, 39.7, 35.4, 28.6, 28.3 (3C), 15.4, 13.9. HRMS (FABþ)
calcd for C36H45N3O7 (m/z) 631.3258; found (m/z) 631.3264.
4.21. Preparation of compound 52
4.19. syn-6-Benzyloxy-3-[8aR-(4-chloro-3-methyl-but-2E-
enyl)-1-methoxy-8S-methyl-4-oxo-3,4,6,7,8,8a-hexahydro-
pyrrolo[1,2-a]pyrazin-3S-ylmethyl]-7-methoxy-indole-1-
carboxylic acid tert-butyl ester (50a)
To NaH (372 mg, 9.29 mmol, 60% dispersion in mineral oil,
freshly washed in pentane) was added a solution of 50a (302 mg,
464 mmol) in dry xylenes (40 mL). This mixture was placed in pre-
heated oil bath (150 ꢁC) and stirred at reflux (135 ꢁC internal
temperature) for 60 min. The reaction mixture (red now) was
cooled in an ice/water bath and was poured into satd NaHCO3 so-
lution (200 mL). After 10 min, the organic phase was separated and
the aqueous phase was extracted with ethyl acetate (3ꢂ100 mL).
The combined organic phases were dried over Na2SO4 and the
solvent was removed in vacuo. As the 1H NMR of the crude material
indicated 10–15% of Boc-deprotected material, the crude reaction
mixture was re-dissolved in CH2Cl2 (5 mL) and triethyl amine
To a solution of 49a (770 mg, 1.22 mmol) in dry DMF (12 mL)
was added 2,4,6-collidine (1.48 g, 12.2 mmol). After 10 min LiCl
(258 mg, 6.09 mmol) was added, after another 10 min MsCl
(419 mg, 3.66 mmol) was added at 0 ꢁC, and the reaction mixture
was stirred for 5 days at room temperature. The reaction mixture
was poured into satd NaHCO3 solution (200 mL), and the aqueous
phase was extracted with ethyl acetate (3ꢂ100 mL). The combined
organic phases were dried over Na2SO4, the solvent was removed in
vacuo, and the residue was purified by flash column chromatog-
raphy (silica, hexane/ethyl acetate: 8:2 then 6:4 then 5:5 then 4:6
then 2:8) to yield 50a (679 mg, 86%) as a foam.
(51.7 mg, 510 mmol) followed by DMAP (56.7 mg, 464 mmol) was
added and the reaction mixture was stirred for 10 min at room
temperature. A solution of Boc2O (304 mg, 1.39 mmol) in dry
CH2Cl2 (1 mL) was added and the stirring continued for 48 h. The
reaction mixture was poured into satd NaHCO3 solution (100 mL),
the organic phase was separated, and the aqueous phase extracted
with ethyl acetate (3ꢂ50 mL). The combined organic phases were
dried over Na2SO4, the solvent was removed in vacuo, and the
residue was purified by flash column chromatography (silica, hex-
ane/ethyl acetate: 9:1 then 8:2 then 7:3 then 6:4 then 5:5) to yield
52 (159 mg, 56%) as a foam. Employing the above conditions
TLC (SiO2, hexanes/EtOAc: 1:4): Rf¼0.37.
25
[a
]
ꢀ4.6 (c 0.32, CHCl3). IR (thin film): 2975, 2942, 2894, 1751,
D
1695, 1652, 1436, 1353, 1266, 1236, 1156, 1026 cmꢀ1
(300 MHz, CDCl3)
.
1H NMR
0.75 (d, J¼7.2 Hz, 3H), 1.63 (s, 9H), 1.69 (s, 3H),
d
1.50–1.70 (m, 2H), 1.95–2.20 (m, 2H), 2.25–2.40 (m, 2H), 2.81 (dd,
J¼9.2, 14.5 Hz, 1H), 3.28 (1/2 ADX, J¼3.0 Hz, JAB¼11.9 Hz), 3.38 (1/2
ADX, J¼3.9 Hz, JAB¼14.6 Hz), 3.66 (s, 3H), 3.91 (s, 3H), 3.95 (s, 2H),
3.90–4.20 (m, 1H), 4.37 (dd, J¼3.9, 8.6 Hz, 1H), 5.17 (s, 2H), 5.34 (t,
J¼7.8 Hz,1H), 6.97 (d, J¼8.6 Hz,1H), 7.22 (d, J¼8.8 Hz,1H), 7.30–7.45
starting with 50b (376 mg, 578 mmol) yielded 52 (273 mg, 77%) as
a foam.
(m, 3H), 7.48 (d, J¼8.9 Hz, 2H). 13C NMR (100 MHz, CDCl3):
d
169.3,
TLC (SiO2, hexanes/EtOAc: 1:1): Rf¼0.31.
25
159.6, 150.2, 149.0, 139.5, 137.9, 135.8, 129.1, 129.1, 128.6 (2C), 127.9,
127.8 (2C), 126.6, 124.1, 117.0, 114.7, 113.8, 83.0, 73.3, 70.3, 61.8, 61.5,
53.0, 51.7, 42.1, 40.0, 35.4, 31.7, 28.3 (3C), 27.7, 16.9, 14.5. HRMS
(FABþ) calcd for C36H44ClN3O6 (m/z) 649.2919; found (m/z)
649.2932.
[
a
]
D
þ21.8 (c 0.45, CHCl3). IR (thin film): 2973, 2940, 2880, 1751,
1682, 1634, 1497, 1417, 1350, 1267, 1235, 1157, 1027, 906 cmꢀ1
NMR (300 MHz, CDCl3)
.
1H
1.33 (d, J¼7.2 Hz, 3H), 1.35–1.45 (m, 1H),
d
1.63 (s, 9H), 1.66 (s, 3H), 2.05–2.20 (m, 2H), 2.20–2.35 (m, 1H), 2.50
(dd, J¼5.2, 10.5 Hz, 1H), 3.09 (1/2 ADX, JAB¼15.9 Hz), 3.30–3.40 (m,
1H), 3.37 (1/2 ADX, JAB¼15.6 Hz), 3.63 (s, 3H), 3.60–3.70 (m, 1H),
3.94 (s, 3H), 4.73 (s, 1H), 4.86 (s, 1H), 5.17 (s, 2H), 6.92 (d, J¼8.5 Hz,
1H), 7.30–7.40 (m, 3H), 7.43 (d, J¼8.4 Hz, 1H), 7.47–7.55 (m, 2H). 13C
4.20. anti-6-Benzyloxy-3-[8aR-(4-chloro-3-methyl-but-2E-
enyl)-1-methoxy-8S-methyl-4-oxo-3,4,6,7,8,8a-hexahydro-
pyrrolo[1,2-a]pyrazin-3R-ylmethyl]-7-methoxy-indole-1-
carboxylic acid tert-butyl ester (50b)
NMR (75 MHz, CDCl3):
d 173.1, 172.2, 149.6, 149.0, 144.3, 139.0,
138.0, 130.5, 128.7 (2C), 128.5, 127.8, 127.7, 127.7 (2C), 116.4, 116.4,
115.2, 113.4, 82.6, 73.4, 68.7, 66.1, 61.5, 54.3, 48.6, 43.1, 40.9, 37.9,
33.1, 28.4 (3C), 27.0, 19.8, 14.2. HRMS (FABþ) calcd for C36H43N3O6
(m/z) 614.3230 [Mþþ1]; found (m/z) 614.3222.
To a solution of 49b (1.26 g, 1.99 mmol) in dry DMF (20 mL) was
added 2,4,6-collidine (2.42 g, 19.9 mmol). After 10 min LiCl
(423 mg, 9.97 mmol) was added, after another 10 min MsCl
(685 mg, 5.98 mmol) was added at 0 ꢁC, and the reaction mixture
was stirred for 5 days at room temperature. The reaction mixture
was poured into satd NaHCO3 solution (200 mL), and the aqueous
phase was extracted with ethyl acetate (3ꢂ100 mL). The combined
organic phases were dried over Na2SO4, the solvent was removed in
vacuo, and the residue was purified by flash column chromatog-
raphy (silica, hexane/ethyl acetate: 10:0 then 9:1 then 8:2 then 7:3
then 6:4) to yield 50b (1.12 g, 86%) as a foam.
4.22. Hexacycle (54)
To Pd(TFA)2 (260 mg, 0.782 mmol) was added acetonitrile
(3.0 mL) and the reaction mixture was stirred for 30 min at room
temperature. Propylene oxide (1.42 g, 24.4 mmol) was added and the
reaction mixture was stirred for 10 min at room temperature and
then this solution was added to a solution of 52 (300 mg,
0.488 mmol) in acetonitrile (2.0 mL). The reaction mixture was stir-
red for 24 h at room temperature, and EtOH (5 mL) was added, fol-
lowed by NaBH4 (92.3 mg, 2.44 mmol) at room temperature. After
3 h, the black reaction mixture was filtered through Celite to remove
palladium black and solvent was removed in vacuo. The residue was
taken in a phosphate buffer solution (pH¼7) and extracted with
TLC (SiO2, hexanes/EtOAc: 1:1): Rf¼0.34.
25
[a
]
þ65.6 (c 0.82, CHCl3). IR (thin film): 2974, 2941, 2894, 1752,
D
1695, 1650, 1436, 1351, 1266, 1235, 1157, 1026 cmꢀ1
(300 MHz, CDCl3)
0.06 (d, J¼6.3 Hz, 3H), 1.25–1.45 (m, 1H), 0.86 (s,
9H), 1.60 (s, 3H), 1.65 (s, 9H), 2.00–2.15 (m, 2H), 2.22 (1/2 ADX,
.
1H NMR
d

K. Sommer, R.M. Williams / Tetrahedron 65 (2009) 3246–3260
3257
EtOAc (3ꢂ100 mL). The combined organic phases were dried over
Na2SO4 and the solvent was removed in vacuo. As the 1H NMR of the
crude material (320 mg) indicated a partly Bn-deprotected product,
the crude reaction mixture was re-dissolved in DMF (5 mL) and
K2CO3 (226 mg, 1.64 mmol) followed by BnBr (224 mg, 1.31 mmol)
was added and the reaction mixture was stirred for 3 days at room
temperature. The reaction mixture was poured into satd NaHCO3
solution (100 mL), the organic phase was separated, and the aqueous
phase extracted with ethyl acetate (3ꢂ50 mL). The combined organic
phases were dried over Na2SO4, the solvent was removed in vacuo,
and the residue was purified by flash column chromatography (silica,
hexane/ethyl acetate: 8:2 then 7:3 then 6:4 then 5:5 then 4:6 then
2:8) to yield 54 (280 mg, 94%) as a foam.
(3C), 20.0, 14.2. HRMS (FABþ) calcd for C36H42DN3O6 (m/z)
614.3215; found (m/z) 614.3212.
4.24. Diketopiperazine (59)
To compound 54 (280 mg, 0.456 mmol) in THF (50 mL) was
added aqueous HCl solution (4.55 mL, 0.1 N) at 0 ꢁC and the re-
action mixture was stirred for 80 min at room temperature. The
reaction mixture was poured into satd NaHCO3 solution (100 mL)
and extracted with EtOAc (3ꢂ100 mL). The combined organic
phases were dried over Na2SO4 and the solvent was removed in
vacuo. The crude reaction product was dissolved in toluene
(20 mL), 2-hydroxypyridine (47.6 mg, 0.501 mmol) was added, and
the reaction mixture was heated under reflux for 3.5 h. The solvent
was removed in vacuo and the residue was purified by flash column
chromatography (silica, hexane/ethyl acetate: 8:2 then 7:3 then 6:4
then 5:5 then 4:6 then 2:8) to yield 59 (222 mg, 81%) as a foam.
TLC (SiO2, hexanes/EtOAc: 2:3): Rf¼0.32.
25
[
a]
þ93.8 (c 0.45, CHCl3). IR (thin film): 2963, 2931, 2874, 1743,
D
1683, 1629, 1501, 1444, 1322, 1246, 1154, 1058, 994 cmꢀ1. 1H NMR
(300 MHz, CDCl3)
d
1.27 (s, 3H), 1.37 (s, 3H), 1.42 (d, J¼7.2 Hz, 3H),
1.60 (s, 9H),1.65–1.80 (m, 2H), 2.05–2.20 (m, 2H), 2.30–2.50 (m, 2H),
3.08 (1/2 ADX, JAB¼17.9 Hz, 1H), 3.20–3.35 (m, 1H), 3.61 (dt, J¼2.4,
5.9 Hz,1H), 3.85 (s, 3H), 3.88 (s, 3H), 3.90 (1/2 ADX, JAB¼16.6 Hz), 5.18
(s, 2H), 6.88 (d, J¼8.9 Hz, 1H), 7.13 (d, J¼8.1 Hz, 1H), 7.30–7.45 (m,
TLC (SiO2, hexanes/EtOAc: 1:4): Rf¼0.40.
25
[a
]
þ97.8 (c 0.5, CHCl3). IR (thin film): 3221, 2970, 2933, 2874,
D
1744, 1692, 1502, 1444, 1329, 1247, 1056 cmꢀ1. 1H NMR (300 MHz,
CDCl3)
d
1.27 (s, 3H), 1.39 (s, 3H), 1.54 (d, J¼7.2 Hz, 3H), 1.60 (s, 9H),
3H), 7.47 (d, J¼7.4 Hz, 2H). 13C NMR (75 MHz, CDCl3):
d
173.5, 172.5,
1.80–1.95 (m, 2H), 2.05–2.20 (m, 1H), 2.25–2.35 (m, 1H), 2.42 (1/2
ADX, J¼11.1 Hz, JAB¼13.9 Hz), 2.54 (1/2 ADX, JAB¼16.8 Hz), 2.59 (1/2
ADX, J¼4.1 Hz, JAB¼11.2 Hz), 3.25–3.35 (m, 1H), 3.60–3.70 (m, 1H),
3.74 (1/2 ADX, JAB¼15.8 Hz), 3.89 (s, 3H), 6.02 (s, 1H), 6.90 (d,
J¼8.5 Hz, 1H), 7.06 (d, J¼8.2 Hz, 1H), 7.30–7.45 (m, 3H), 7.47 (d,
153.7, 147.8, 139.8, 137.7, 136.9, 131.1, 128.5 (2C), 127.8, 127.6 (2C),
124.7, 113.6, 111.7, 110.6, 84.3, 72.9, 66.5, 65.1, 60.8, 54.3, 49.2, 43.0,
41.2, 37.1, 33.1, 33.0, 28.1, 27.6 (3C), 27.0, 21.0,14.2. HRMS (FABþ) calcd
for C36H43N3O6 (m/z) 613.3152; found (m/z) 613.3147.
J¼7.5 Hz, 1H). 13C NMR (75 MHz, CDCl3):
d 174.0, 169.6, 153.4, 148.0,
4.23. Deuterium-labeled hexacycle (56)
139.8, 137.6, 136.9, 131.1, 128.6 (2C), 127.9, 127.6 (2C), 124.0, 113.3,
111.8, 108.3, 84.7, 72.8, 67.1, 60.9, 59.5, 51.1, 43.5, 41.9, 36.7, 32.8,
31.8, 27.7, 26.7 (3C), 25.1, 20.6, 13.4. HRMS (FABþ) calcd for
C35H41N3O6 (m/z) 599.2995; found (m/z) 599.3005.
To Pd(TFA)2 (203 mg, 0.611 mmol) was added acetonitrile
(2.0 mL) and the reaction mixture was stirred for 15 min at room
temperature. Propylene oxide (1.18 g, 20.4 mmol) was added and
the reaction mixture was stirred for 10 min at room temperature
and then this solution was added to a solution of 52 (250 mg,
0.407 mmol) in acetonitrile (2.0 mL). The reaction mixture was
stirred for 18 h at room temperature, and EtOD (4 mL) was added,
followed by NaBD4 (171 mg, 4.07 mmol) at room temperature. After
8 h, the black reaction mixture was filtered through Celite to
remove palladium black and the solvent was removed in vacuo. The
residue was taken in satd NaHCO3 solution (100 mL), and extracted
with EtOAc (3ꢂ100 mL). The combined organic phases were dried
over Na2SO4 and the solvent was removed in vacuo. As the 1H NMR
of the crude material indicated a partly Bn-deprotected product,
the crude reaction mixture was re-dissolved in DMF (4 mL) and
K2CO3 (169 mg, 1.22 mmol) followed by BnBr (167 mg, 0.98 mmol)
was added and the reaction mixture was stirred for 2 days at room
temperature. The reaction mixture was poured into satd NaHCO3
solution (100 mL), the organic phase was separated, and the
aqueous phase extracted with ethyl acetate (3ꢂ50 mL). The
combined organic phases were dried over Na2SO4, the solvent was
removed in vacuo, and the residue was purified by flash column
chromatography (silica, hexane/ethyl acetate: 8:2 then 7:3 then 6:4
then 5:5 then 4:6 then 2:8) to yield 56 (157 mg, 63%) as a foam.
4.25. Diketopiperazine (60)
To compound 56 (160 mg, 0.260 mmol) in THF (24 mL) was
added aqueous HCl solution (2.60 mL, 0.1 N) at 0 ꢁC and the re-
action mixture was stirred for 85 min at room temperature. The
reaction mixture was poured into satd NaHCO3 solution (100 mL)
and extracted with EtOAc (3ꢂ100 mL). The combined organic
phases were dried over Na2SO4 and the solvent was removed in
vacuo. The crude reaction product was dissolved in toluene (5 mL),
2-HO-py (27.2 mg, 0.286 mmol) was added, and the reaction mix-
ture was heated under reflux for 5 h. The solvent was removed in
vacuo and the residue was purified by flash column chromatogra-
phy (silica, hexane/ethyl acetate: 8:2 then 7:3 then 6:4 then 5:5
then 4:6 then 2:8) to yield 60 (135 mg, 86%) as a foam.
TLC (SiO2, hexanes/EtOAc: 1:4): Rf¼0.41.
25
[a]
þ104 (c 0.55, CHCl3). IR (thin film): 3228, 2973, 2934, 2877,
D
1744, 1692, 1502, 1455, 1369, 1246, 1153, 1055 cmꢀ1
(300 MHz, CDCl3)
.
1H NMR
1.28 (s, 3H), 1.38 (d, J¼5.9 Hz, 2H), 1.54 (d,
d
J¼7.1 Hz, 3H),1.60 (s, 9H),1.80–1.95 (m, 2H), 2.05–2.20 (m,1H), 2.25–
2.35 (m, 1H), 2.42 (1/2 ADX, J¼10.4 Hz, JAB¼13.5 Hz), 2.54 (1/2 ADX,
JAB¼15.8 Hz), 2.60 (1/2 ADX, J¼4.4 Hz, JAB¼10.3 Hz), 3.25–3.35 (m,
1H), 3.60–3.70 (m, 1H), 3.73 (1/2 ADX, JAB¼15.6 Hz, 1H), 3.89 (s, 3H),
5.17 (s, 2H), 5.97 (s, 1H), 6.90 (d, J¼8.5 Hz,1H), 7.06 (d, J¼8.2 Hz,1H),
7.30–7.45 (m, 3H), 7.47 (d, J¼6.8 Hz, 2H). 13C NMR (75 MHz, CDCl3):
TLC (SiO2, hexanes/EtOAc: 2:3): Rf¼0.34.
25
[
a]
þ117.3 (c 0.15, CHCl3). IR (thin film): 2973, 2936, 2877, 1742,
D
1683, 1634, 1505, 1456, 1324, 1245, 1152, 1056 cmꢀ1
(400 MHz, CDCl3)
.
1H NMR
1.27 (s, 3H), 1.36 (d, J¼8.1 Hz, 2H), 1.42 (d,
d
d 174.0, 169.6, 153.4, 148.0, 139.8, 137.6, 136.9, 131.1, 128.6 (2C), 127.9,
J¼7.2 Hz, 3H), 1.56 (s, 9H), 1.55–1.65 (m, 2H), 1.65–1.70 (m, 2H),
2.05–2.15 (m, 2H), 2.29 (dd, J¼5.2, 11.1 Hz), 2.30–2.45 (m, 1H), 3.00
(1/2 ADX, JAB¼16.4 Hz, 1H), 3.20–3.35 (m, 1H), 3.61 (dt, J¼2.2,
7.9 Hz, 1H), 3.78 (s, 3H), 3.88 (s, 3H), 3.89 (1/2 ADX, JAB¼16.2 Hz),
5.18 (s, 2H), 6.88 (d, J¼8.6 Hz, 1H), 7.13 (d, J¼8.5 Hz, 1H), 7.32 (t,
J¼7.5 Hz, 1H), 7.39 (t, J¼7.1 Hz, 2H), 7.47 (d, J¼6.8 Hz, 2H). 13C NMR
127.6 (2C), 124.0, 113.3, 111.9, 108.3, 84.7, 72.9, 67.2, 60.9, 59.5, 51.1,
43.5, 41.9, 36.6, 32.8, 31.8, 27.7(3C), 26.7, 25.1, 20.6,13.4. HRMS (FABþ)
calcd for C35H40DN3O6 (m/z) 600.3058; found (m/z) 600.3070.
4.26. Deuterium-labeled monoketopiperzine (62)
(100 MHz, CDCl3):
d
173.8, 172.7, 153.9, 148.0, 140.0, 137.9, 137.0,
To a suspension of LiAlD4 (2.77 g, 66.0 mmol) in dry heptanes
was added triisobutylaluminum (11.9 g, 60.6 mmol) at room
temperature under argon. The reaction mixture was heated at
131.3, 128.7 (2C), 128.0, 127.7 (2C), 124.8, 113.7, 111.8, 110.8, 84.4,
72.9, 66.5, 65.2, 60.9, 54.4, 49.2, 43.0, 41.3, 37.1, 33.1, 33.0, 28.1, 27.6

3258
K. Sommer, R.M. Williams / Tetrahedron 65 (2009) 3246–3260
100 ꢁC for 6 h. After cooling to room temperature, the resulting
suspension was filtered and the insoluble gray solid was washed
with heptanes. The solvent was removed in vacuo and the liquid
residue was re-dissolved in dry toluene to give 1.3 M DIBAL-D
solution in toluene. Compound 60 (342 mg, 0.569 mmol) was
dissolved in toluene (11.0 mL) and cooled to ꢀ78 ꢁC. At this
temperature DIBAL-D solution in toluene (1.75 mL, 1.3 M) was
added dropwise. The reaction mixture was allowed to warm to
room temperature slowly and was stirred for 4 days at room
temperature. Satd NaHCO3 solution (100 mL), followed by satd
Na–K-tartrate solution (100 mL) was added and the reaction
mixture was stirred for 1 h. The organic phase was separated and
the aqueous phase was extracted with ethyl acetate (3ꢂ200 mL).
The combined organic phases were dried over Na2SO4 and the
solvent was removed in vacuo. The residue (mixture of 70%
product and 30% substrate, 0.35 g) was re-dissolved in toluene
(11.0 mL) and cooled to ꢀ78 ꢁC. At this temperature, DIBAL-D
solution in toluene (1.33 mL, 1.3 M) was added dropwise. The re-
action mixture was allowed to warm to room temperature slowly
and was stirred for 4 days at room temperature. Additional DIBAL-
D solution in toluene (0.3 mL, 1.3 M) was added dropwise, and the
reaction mixture was stirred for 2 days at room temperature. Satd
NaHCO3 solution (100 mL), followed by satd Na–K-tartrate solu-
tion (100 mL) was added, and the reaction mixture was stirred for
1 h. The organic phase was separated and the aqueous phase was
extracted with ethyl acetate (3ꢂ200 mL). The combined organic
phases were dried over Na2SO4 and the solvent was removed in
vacuo. The residue (0.35 g) was purified by flash column chro-
matography (silica, hexane/ethyl acetate: 8:2 then 7:3 then 6:4
then 5:5 then 4:6 then 2:8) and (silica, CH2Cl2/MeOH: 10:0 then
99:1 then 98:2 then 97:3 then 96:4 then 95:5) to give 62 together
with 15% of the Boc-deprotected analogue as a non-separable
mixture: (189 mg, 52%).
silica, CH2Cl2/MeOH: 95:5) to give 64, together with 15% of the Boc-
deprotected analogue as a non-separable mixture: (63 mg, 64%).
TLC (SiO2, CH2Cl2/MeOH: 95:5): Rf¼0.38.
IR (thin film): 3354, 3195, 2933, 2873, 2841, 1748, 1673, 1494,
1447, 1370, 1249, 1153, 1023 cmꢀ1. 1H NMR (400 MHz, CDCl3)
d 1.39
(d, J¼8.7 Hz, 2H), 1.41 (s, 3H), 1.57 (d, J¼9.4 Hz, 3H), 1.59 (s, 9H),
1.50–1.65 (m, 2H), 1.70 (d, J¼10.2 Hz, 1H), 1.85–2.10 (m, 3H), 2.10–
2.25 (m, 2H), 2.22 (dt, J¼4.4, 10.9 Hz, 1H), 2.79 (1/2 ADX,
JAB¼15.8 Hz, 1H), 2.83 (1/2 ADX, JAB¼15.1 Hz), 3.17 (s, 3H), 3.15–3.30
(m, 1H), 3.93 (s, 3H), 6.71 (s, 1H), 7.12 (d, J¼8.5 Hz, 1H), 7.16 (d,
J¼8.2 Hz, 1H). 13C NMR (100 MHz, CDCl3):
d 174.3, 152.8, 143.6,
139.0, 138.4, 130.5, 128.5, 118.2, 113.7, 108.8, 85.5, 65.6, 61.9, 55.8,
55.7, 53.9, 53.8, 47.9, 40.5, 37.4, 35.7, 30.7, 30.3, 29.9, 27.6 (3C), 21.9,
13.2. HRMS (FABþ) calcd for C29H36D3N3O7S (m/z) 577.2775
[Mþþ1]; found (m/z) 577.2762.
4.28. Indoline (65)
A solution of 64 (63 mg, 109 mmol) in dry methanol (6 mL) was
stirred under H2 (1 atm) for 3 days at room temperature in the
presence of Pd(OH)2/C (20 wt %) (1.2 g). The catalyst was removed
by filtration through a pad of Celite, which was washed with
methanol (300 mL), and the solvent was removed in vacuo to yield
65 (25 mg, 47%) as a white solid.
TLC (SiO2, CH2Cl2/MeOH: 95:5): Rf¼0.21.
IR (thin film): 3180, 3057, 2950, 2929, 2874, 1690, 1673, 1485,
1454, 1368, 1276, 1167 cmꢀ1. 1H NMR (400 MHz, CD3OD)
d 0.48 (s,
3H), 1.02 (d, J¼7.3 Hz, 2H), 1.29 (d, J¼6.8 Hz, 3H), 1.25–1.35 (m, 2H),
1.37 (d, J¼5.9, 13.2 Hz, 1H), 1.48 (s, 9H), 1.64 (dd, J¼5.6, 11.1 Hz, 1H),
1.72 (dt, J¼4.4,10.4 Hz,1H),1.75–1.85 (m,1H),1.85–1.95 (m,1H),1.98
(1/2 ADX, JAB¼11.5 Hz, 1H), 2.00–2.15 (m, 3H), 2.38 (1/2 ADX,
JAB¼14.7 Hz), 3.03 (dt, J¼4.0, 9.7 Hz, 1H), 3.78 (t, J¼7.4 Hz, 1H), 3.84
(s, 3H), 4.30 (d, J¼5.5 Hz,1H), 6.75 (d, J¼7.2 Hz,1H), 6.85 (d, J¼8.4 Hz,
TLC (SiO2, hexanes/EtOAc: 3:7): Rf¼0.41.
1H), 7.08 (t, J¼8.3 Hz,1H).13C NMR (100 MHz, CD3OD):
d 175.1,156.2,
25
[a
]
þ41.2 (c 0.50, CHCl3). IR (thin film): 3196, 2947, 2930, 2870,
151.7, 140.3, 133.7, 127.4, 114.9, 112.5, 82.0, 74.2, 67.2, 56.4, 56.1, 54.8,
47.1, 41.8, 41.4, 38.8, 31.5, 31.3, 30.9, 28.7 (3C),17.3,13.5. HRMS (FABþ)
calcd for C28H36D3N3O4 (m/z) 484.3129; found (m/z) 484.3112.
D
1746, 1674, 1501, 1455, 1369, 1245, 1153, 1055 cmꢀ1
(400 MHz, CDCl3)
.
1H NMR
1.39 (d, J¼10.3 Hz, 2H), 1.42 (d, J¼6.4 Hz, 3H),
d
1.56 (s, 3H), 1.62 (s, 9H), 1.60–1.75 (m, 2H), 1.85–1.95 (m, 1H), 1.95–
2.10 (m, 2H), 2.10–2.25 (m, 2H), 2.30 (dt, J¼4.5, 13.9 Hz, 1H), 2.74 (1/
2 ADX, JAB¼17.4 Hz, 1H), 2.81 (1/2 ADX, JAB¼16.1 Hz), 3.15–3.30 (m,
1H), 3.92 (s, 3H), 5.17 (s, 2H), 6.09 (s, 1H), 6.90 (d, J¼7.5 Hz, 1H), 7.03
(d, J¼8.4 Hz, 1H), 7.33 (t, J¼7.0 Hz, 1H), 7.39 (t, J¼7.3 Hz, 2H), 7.47 (d,
4.29. Deprotected indoline (66)
To 65 (32 mg, 66.3 mmol)inCH2Cl2 (1.5 mL) was added TFA (605 mg,
5.30 mmol) at 0 ꢁC and the reaction mixture was stirred for 3 h at 0 ꢁC.
The solvent was removed in vacuo and the residue taken in NaHCO3/
EtOAc (10 mL), the organic phase was separated, and the aqueous
(pH¼8.5) was extracted with EtOAc (3ꢂ5 mL). The combined organic
phases were dried over Na2SO4, the solvent was removed in vacuo, and
the residue was purified by preparative thin layer chromatography
(2 mm/silica, CH2Cl2/MeOH: 9:1) to give 66 (15.5 mg, 98%).
J¼7.2 Hz, 2H). 13C NMR (100 MHz, CDCl3):
d 174.4, 153.6, 148.0,
141.4, 137.7, 137.0, 131.4, 128.7 (2C), 128.0, 127.6 (2C), 124.1, 112.7,
111.9, 108.1, 84.8, 72.8, 65.6, 60.8, 55.8, 53.8, 47.9, 40.5, 35.6, 30.7,
30.2, 30.0, 28.7, 28.4, 27.6 (3C), 22.0, 13.3. HRMS (FABþ) calcd for
C35H40D3N3O5 (m/z) 588.3391; found (m/z) 588.3412.
4.27. Mesylate (64)
TLC (SiO2, CH2Cl2/MeOH: 9:1): Rf¼0.52.
IR (thin film): 3283, 3179, 3049, 2945, 2930, 2874, 1668, 1490,
A solution of 62 (100 mg, 170
mmol) in dry ethanol (6 mL) was
1463, 1369, 1261, 1117 cmꢀ1. 1H NMR (400 MHz, CD3OD)
d 0.81 (s,
stirred under H2 (1 atm) for 19 h at room temperature in the
presence of Pd/C (10 wt %) (50 mg). The catalyst was removed by
filtration through a pad of Celite, which was washed with ethanol
(30 mL), and the solvent was removed in vacuo to yield the
deprotected intermediate (85 mg,100%) as a white solid, which was
used further without purification. TLC (SiO2, CH2Cl2/MeOH: 9:1):
Rf¼0.46. To a solution of the deprotected intermediate (85 mg,
3H), 0.96 (d, J¼7.2 Hz, 2H), 1.32 (d, J¼6.8 Hz, 3H), 1.38 (dd, J¼7.9,
13.0 Hz, 2H), 1.63 (dd, J¼7.9, 10.9 Hz, 1H),1.72 (dq, J¼4.3, 10.9 Hz,
1H), 1.70–1.80 (m, 1H), 1.90–2.05 (m, 4H), 2.10–2.25 (m, 2H), 3.07
(dt, J¼4.3, 9.2 Hz, 1H), 3.49 (dd, J¼7.5, 8.3 Hz, 1H), 3.60 (d, J¼8.7 Hz,
1H), 3.79 (s, 3H), 6.55–6.75 (m, 3H). 13C NMR (100 MHz, CD3OD):
d
175.4, 146.5, 141.5, 133.8, 120.3, 116.5, 111.1, 71.0, 67.8, 56.8, 56.7,
56.0, 54.3, 41.3, 41.3, 37.4, 32.7, 31.3, 30.8, 17.8, 13.6. HRMS (FABþ)
170
mmol) in CH2Cl2 (2.0 mL) was added pyridine (2.0 mL) followed
calcd for C23H28D3N3O2 (m/z) 384.2605; found (m/z) 384.2636.
by MsCl (195 mg, 1.70 mmol) at 0 ꢁC and the reaction mixture was
stirred at room temperature for 12 h. The solvent was removed in
vacuo and the residue taken in NaHCO3/EtOAc (100 mL), the or-
ganic phase was separated and the aqueous (pH¼8.5) was extracted
with EtOAc (3ꢂ50 mL). The combined organic phases were dried
over Na2SO4, the solvent was removed in vacuo, and the residue
was purified by preparative thin layer chromatography (2 mm/
4.30. 7-HO-Pre-paraherquamide (27)
To 66 (15.5 mg, 40 mmol) in CH2Cl2 (0.5 mL) was added BBr3
(150
m
L, 1 M in CH2Cl2) at ꢀ78 ꢁC and the reaction mixture was
allowed to warm to room temperature slowly over 13 h. The re-
action mixture was cooled to ꢀ78 ꢁC and MeOH (0.5 mL) was

K. Sommer, R.M. Williams / Tetrahedron 65 (2009) 3246–3260
3259
added. The reaction mixture was warmed to room temperature and
the solvent was removed in vacuo. The HBr-salt of (20) was taken in
NaHCO3/EtOAc (10 mL), the organic phase was separated, and the
aqueous (pH¼8.5) was extracted with EtOAc (3ꢂ5 mL). The
combined organic phases were dried over Na2SO4, the solvent was
removed in vacuo, and the residue was purified by preparative thin
layer chromatography (2 mm/silica, CH2Cl2/MeOH: 9:1) to give
a mixture of 67 (15 mg, 87%) and 27 (4:1). The mixture was dis-
solved in EtOAc, satd NaHCO3 solution was added, and the biphasic
mixture was stirred under air atmosphere and at room temperature
for 48 h. The organic phase was separated, the aqueous phase was
extracted with EtOAc, and the combined organic phases were dried
over Na2SO4. The solvent was removed in vacuo and the residue
was purified by preparative thin layer chromatography (2 mm/sil-
ica, CH2Cl2/MeOH: 95:5) to give 27 (14.5 mg, >99%).
CHCl3 and shaken at room temperature for an additional 24 h. The
Celite and mycelia suspension was filtered through Whatman #2
paper. The organic solvent from the combined filtrates was con-
centrated by rotary evaporation. The aqueous solution from the
feeding experiment was combined with aqueous residue and the
mixture was acidified to pH 3 with glacial acetic acid. The acidic
solution was filtered through a pad of Celite and extracted with
ethyl acetate (400 mLꢂ3). The aqueous layer was made basic, pH
12, by the addition of 10% aqueous Na2CO3. The aqueous layer was
extracted with ethyl acetate (450 mLꢂ4). The combined organic
extracts from the basic extraction were washed with brine
(800 mL), dried (Na2SO4), filtered, and concentrated by rotary
evaporation (34 mg). The paraherquamide A was purified by pre-
parative thin layer chromatography with 95:5 CH2Cl2/MeOH to give
14.5 mg of the natural product.
TLC (SiO2, CH2Cl2/MeOH: 95:5): Rf¼0.20.
25
[a]
ꢀ26.7(c0.038,MeOH/CH2Cl2:1:1). IR(thinfilm):3355, 3196,
D
4.32. Examination of percent deuterium incorporation in the
isolated sample of paraherquamide A by 2H NMR
2950, 2921, 2850, 1662, 1456, 1374, 1260, 1113 cmꢀ1
(400 MHz, CD3OD/CDCl3: 5:1)
.
1H NMR
1.36 (d, J¼7.1 Hz, 3H), 1.41 (d,
d
J¼6.8 Hz, 2H),1.42 (s, 3H),1.74 (1/2 ADX, J¼10.7 Hz, JAB¼18.8 Hz,1H),
1.86 (dt, J¼4.7, 10.2 Hz, 1H), 1.95–2.10 (m, 2H), 2.17 (1/2 ADX,
J¼11.9 Hz, JAB¼17.5 Hz,1H), 2.10–2.25 (m, 1H), 2.29 (dt, J¼4.9, 9.4 Hz,
1H), 2.87 (s, 2H), 3.20 (m,1H), 6.54 (d, J¼7.3 Hz,1H), 6.84 (t, J¼7.7 Hz,
1H), 6.90 (d, J¼6.7 Hz, 1H). 13C NMR (100 MHz, CD3OD/CDCl3: 5:1):
Examination of percent deuterium incorporation in the isolated
sample of paraherquamide A (14 mg) was accomplished by comparing
the signal intensities from the partially enriched sample with the signal
intensity from the solvent, CHCl3, that was spiked with a known amount
of 99.8% CDCl3. The spiked solventwasmadeup asfollows. CDCl3 (100 mL,
d
175.9, 143.8, 141.6, 129.9, 127.5, 120.3, 110.2, 107.0, 105.1, 66.4, 57.6,
99.8%)wasadded to 4.90 mL of CHCl3. This produced a solvent with 2% by
volume of CDCl3. MeOH (5 mL) and 90 mL CHCl3 were added. This pro-
duced a solvent with 0.1% by volume of CDCl3. Several small aliquots of
this thoroughly mixed solvent were added to a round bottom flask
containing the sample, swirled around and then decanted into a new
Wilmad 535pp NMR tube. The sample volume in the NMR tube was
57.5, 54.7, 47.4, 41.3, 35.1, 31.3, 30.6, 30.6, 24.4,13.6.HRMS(FABþ)calcd
for C22H24D3N3O2 (m/z) 369.2370 [Mþþ1]; found (m/z) 369.2370.
4.31. Feeding experiment with 7-HO-pre-paraherquamide in
P. fellutanum
brought finally to 675 mL by adding the mixed solvent dropwise until the
Each malt extract agar slant was streaked with P. fellutanum
volume mark on the NMR tube was reached. The NMR signal averaging
transients were acquired at a rate that allowed the steady-state CDCl3
magnetization to recover to 91% of its value. This was done to acquire
more transients for better signal averaging of the deuterium that are
incorporated in the very dilutesamplesincetheir T1s are almostcertainly
much smaller than those of CDCl3. This means that the signal intensity
from the CDCl3 is slightly underestimated and should be corrected by this
factor before comparing directly with that from the deuterium in-
corporated in the sample. There are several sources of error in this
method. It is estimated that the error in the volumetric measurements is
about 1% overall. It is assumed that T1s for the sample are much shorter
than that for the CDCl3 necessitating that the sample was dissolved al-
though it is assumed that nearly all of the sample was dissolved in the
mixed solvent. The CHCl3 used as the base for the mixed solvent has an
unknown amount of deuterium incorporated although it is likely no
more than what is naturally abundant at a level of 0.012%. This could be
tested byNMR but is averysmall sourceof error. The spectral integrations
plotted assume 20,000 ppm by volume, with no correction for T1 ap-
plied, of deuterium for the CDCl3 signal in the mixed solvent. The values
that are finally reported will need to first take the steady-state value of
the CDCl3 integral into account. The NMR data was acquired on Varian
Inova-500 NMR equipped with a 5 mm broadband tunable probe. The
probe frequency was set to 76.78 MHz and the lock channel was de-
termined. The sample was shimmed in the magnet with the lock off (free
drift) and by using 1H PFG shimming. The NMR signal was acquired with
spores (50 mL of a 10% glycerol/water suspension). Malt extract agar
(500 mL) is prepared by combining malt extract (10 g), dextrose
(10 g), peptone (0.5 g), and agar (10 g) in 500 mL distilled water and
heating gently until all solids dissolve. The slants were then ster-
ilized in an autoclave at 250 ꢁC for 45 min. After inoculation, the
slants were incubated in the dark at 25 ꢁC for 7–14 days. The spores
of two slants were shaken into a flask containing 400 mL sterile
corn steep liquor broth. One liter corn steep liquor broth is prepared
by combining corn steep liquor (22 g) with dextrose (40.0 g) and
heating gently until all solids dissolve. The broth was then sterilized
in an autoclave at 250 ꢁC for 45 min. The inoculated flask was in-
cubated in the dark at 25 ꢁC for 7 days. The broth was decanted
from the flask leaving a disk of P. fellutanum. A sterile solution of 7-
HO-pre-paraherquamide (27) in trace element solution (7 mg in
100 mL) was added by dripping it down the side of the flask and
under the mycelia using a sterile syringe. A detergent had to be
used to dissolve the water-insoluble proposed precursor. The
compound was dissolved in a 10% solution of absolute ethanol/
chloroform. TWEEN 80 (0.1) was added to the dissolved precursor.
The solvent was removed in vacuo, trace element solution was
added, and the solution filtered through sterile antibacterial filter.
The flask was left in the incubator another 14 days. The flasks were
swirled daily to allow even distribution of the labeled compound.
The aqueous solution containing the precursor was decanted off.
Methylene chloride (1–2 mL) was added to the solution, which was
then covered and stored at 4 ꢁC. The mycelia were pureed with
500 mL 1:1 MeOH/CHCl3 in an Oster blender. The contents of the
blender were poured into a 2 L Erlenmeyer flask. The blender was
rinsed three times with 1:1 MeOH/CHCl3 and the suspension of
mycelia cells was diluted to a volume of 1.2 L with 1:1 MeOH/CHCl3.
The mycelia cells were placed in a shaker at room temperature for
24 h. Celite (30 g) was added to the flask. The suspension was fil-
tered through Whatman #2 paper. The filtrate was stored at 4 ꢁC.
The residual Celite and mycelia was suspended in 1.2 L 1:1 MeOH/
the following parameters: 25 ꢁC, 8.8 s RF pulse (90ꢁ tip angle), 1500 Hz
m
spectral window, 2K complex data points, total recycle time of 2.8 s,
Gaussian smoothing, spectral complex transform 16K. The main magnet
field drifts naturallyless than 0.1 Hz perhour. Total acquisition time:40 h.
Acknowledgements
Financial support from the National Institutes of Health (Grant
CA070375) is gratefully acknowledged. K.M.S. acknowledges the

3260
K. Sommer, R.M. Williams / Tetrahedron 65 (2009) 3246–3260
15. Lee, B. H.; Clothier, M. F.; Dutton, F. E.; Nelson, S. J.; Johnson, S. S.; Thompson,
D. P.; Geary, T. G.; Whaley, H. D.; Haber, Ch. L.; Marshall, V. P.; Kornis, G. I.;
McNally, P. L.; Ciadella, J. I.; Martin, D. G.; Bowman, J. W.; Baker, C. A.; Coscarelli,
E. M.; Alexander-Bowman, S. J.; Davis, J. P.; Zinser, E. W.; Wiley, V.; Lipton, M. F.;
Mauragis, M. A. Curr. Top. Med. Chem. 2002, 2, 779.
16. Lee, B. H. J. Labelled Compd. Radiopharm. 2002, 45, 1209.
17. (a) Wulff, J. E.; Herzon, S. B.; Siegrist, R.; Myers, A. G. J. Am. Chem. Soc. 2007,
129, 4898; (b) Wulff, J. E.; Siegrist, R.; Myers, A. G. J. Am. Chem. Soc. 2007,
129, 14444.
Deutsche Forschungsgemeinschaft Postdoctoral Fellowship (SO
507/1-2). We wish to thank Dr. Kenny Miller for help with the
preparation of this manuscript. This paper is dedicated to Professor
Justin Dubois, of Stanford University for receiving the Tetrahedron
Young Investigator Award.
References and notes
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