Total synthesis, stereochemical assignment, and biological activity of all known (-)-trigonoliimines

Article abstract of DOI:10.1021/jo4020358

A full account of our concise and enantioselective total syntheses of all known (-)-trigonoliimine alkaloids is described. Our retrobiosynthetic analysis of these natural products enabled identification of a single bistryptamine precursor as a precursor to all known trigonoliimines through a sequence of transformations involving asymmetric oxidation and reorganization. Our enantioselective syntheses of these alkaloids enabled the revision of the absolute stereochemistry of (-)-trigonoliimines A, B, and C. We report that trigonoliimines A, B, C and structurally related compounds showed weak anticancer activities against HeLa and U-937 cells.

Full text of DOI:10.1021/jo4020358

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Total Synthesis, Stereochemical Assignment, and Biological Activity
of All Known (−)-Trigonoliimines
Sunkyu Han,† Karen C. Morrison,‡ Paul J. Hergenrother,‡ and Mohammad Movassaghi*,†
†Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
‡Department of Chemistry, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States
S
* Supporting Information
ABSTRACT: A full account of our concise and enantiose-
lective total syntheses of all known (−)-trigonoliimine
alkaloids is described. Our retrobiosynthetic analysis of these
natural products enabled identification of a single bistrypt-
amine precursor as a precursor to all known trigonoliimines
through a sequence of transformations involving asymmetric
oxidation and reorganization. Our enantioselective syntheses
of these alkaloids enabled the revision of the absolute
stereochemistry of (−)-trigonoliimines A, B, and C. We report
that trigonoliimines A, B, C and structurally related
compounds showed weak anticancer activities against HeLa
and U-937 cells.
of ( )-trigonoliimines A and B using carbanion mediated
seven-membered ring formation as a key step.11 In 2012, Zhu
and co-workers reported the total synthesis of ( )-trigonolii-
mine B using a late-stage Bischler−Napieralski reaction.12 This
strategy was also applied in Hao and co-workers’ synthesis of
the hexacyclic skeleton of ( )-trigonoliimines A and B.13 Most
recently, Hao and co-workers reported the total synthesis of
( )-trigonoliimine A using a Strecker reaction for the
formation of the C20 tetrasubstituted carbon center and a
Houben−Hoesch14 type reaction for the seven-membered ring
forming cyclization.15 Herein, we detail the full account of our
synthetic approach to the trigonoliimine alkaloids, which
currently remains the only solution to access all known
trigonoliimines in enantiomerically enriched form. We also
report anticancer activities of trigonoliimines A−C and
structurally related alkaloids against HeLa (human cervical
cancer) and U-937 (human lymphoma) cell lines.
INTRODUCTION
■
Bisindole alkaloids containing 2,2′-connectivity constitute a
large number of natural products which includes fascaplycins
and indolocarbazole glycopeptides.1,2 The potent antitumor
activity of indolocarbazole glycopeptides such as rebeccamycin3
and stauroporine4 have stimulated the development of
synthetic analogs of this family of natural products for the
development of anticancer agents.5 Additionally, recent studies
have shown possible application of these natural products as
selective and sensitive chemical sensors based on their unique
chemiluminescence properties.6
In 2010, Hao and co-workers reported the isolation of 2,2′-
linked bistryptamine alkaloids (+)-trigonoliimines A (1) and B
(2) along with (−)-trigonoliimine C (3) from the leaves of
Trigonostemon lii Y. T. Chang collected in the Yunnan province
of China.7 From their biological studies of these alkaloids, 1
showed modest activity in an anti-HIV assay (EC50 = 0.95 μg/
mL, TI = 7.9). Fascinated by their unique molecular
architecture, we initiated a synthetic program aimed to provide
a general solution to trigonoliimine alkaloids in enantiomeri-
cally enriched form. In 2011, we completed the first total
synthesis of all known (−)-trigonoliimines A (1), B (2), and C
(3) using a synthetic strategy based on asymmetric oxidation
and reorganization of a single heterodimeric bistryptamine and
revised the absolute stereochemistry of all (−)-trigonoliimines.8
In a contemporaneous study, Tambar and co-workers reported
the total synthesis of ( )-trigonoliimine C,9 and shortly after,
Hao and co-workers published the synthesis of the pentacyclic
skeleton of ( )-trigonoliimine C using a similar oxidation and
1,2-alkyl shift strategy.10 Interest toward these fascinating
natural products has continued and Shi and co-workers
reported the synthesis of the desmethoxy hexacyclic skeleton
RESULTS AND DISCUSSION
■
Design Plan for the Total Synthesis. Oxidation and
rearrangement of 2,3-disubstituted indoles has served as an
efficient strategy to access the indoxyl moiety and has been
applied in the total synthesis of various alkaloids.16 In 2008, our
laboratory proposed a synthetic strategy to access calycantha-
ceous alkaloids through oxidation and rearrangement of a 2,2′-
bistryptamine derivative.17,18 Our continued interest in this area
yielded an efficient and stereocontrolled synthetic strategy
involving oxidation and rearrangement of 2,3-disubstituted
indoles, especially those with an aryl substituent at the 2-
Received: September 12, 2013
Published: October 15, 2013
© 2013 American Chemical Society
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position, for efficient access to oxindole products.19 Despite
outstanding advancements in the area of asymmetric
oxidation,20 enantioselective oxidation of 2,3-disubstituted
indoles remains a challenging problem.21,22 Recently, in
collaboration with the Miller group, we reported an asymmetric
oxidation of 2,3-disubstituted indole for the formation of
enantiomerically enriched hydroxyindolenines using an aspartyl
based peptide catalyst.23,24
Scheme 1. Retrosynthetic Analysis of (−)-Trigonoliimines
A−C (1−3) and Isotrigonoliimine C (4)
With these considerations in mind, the molecular structure of
trigonoliimine alkaloids (Figure 1) drew our immediate
Figure 1. Structure of (−)-trigonoliimines A−C (1−3) with revised
absolute stereochemistry (ref 8) and other representative trigonoste-
mon alkaloids.
attention upon their isolation due to their possible structural
relevance to intermediates in our primary investigations toward
chimonanthine (S6, Scheme S1, Supporting Information).17
Our unified strategy for the enantioselective total synthesis of
all known trigonoliimines was based on the hypothesis that
bistryptamine heterodimer 15 may serve as a common
biosynthetic precursor to these alkaloids (Scheme 1). We
envisioned that mono-oxidation of bistryptamine 15 would
generate hydroxyindolenines 9 and 10, intermediates that could
serve as a branching point from which to access the two distinct
molecular architectures present in the trigonoliimine alkaloid
family (Scheme 1).
To assemble the amidine moiety present in trigonoliimines A
(1) and B (2), we designed a late stage N-formylation and
intramolecular condensation of azepanes 5 and 6. We planned
to access azepanes 5 and 6 from aminals 7 and 8 via ionization
and stereoretentive intramolecular trapping of carbinol C20 by
N12 and subsequent rearrangement. The requisite cis-fused
aminals 7 and 8 could result from intramolecular cyclization of
hydroxyindolenines 9 and 10, respectively. Retrosynthetic
analysis of trigonoliimine C (3) and isotrigonoliimine C (4)
commenced with formylations of N24 in imines 11 and 12.
The seven-membered imine moiety would be derived from an
intramolecular condensation of N12 to C15 of indoxyls 13 and
14, respectively. Indoxyls 13 and 14 would be accessible via a
stereospecific Wagner−Meerwein type 1,2-alkyl shift25 of
hydroxyindolenines 9 and 10, the common intermediates that
serve as gateways to access all trigonoliimines.
at the stereogenic centers of (+)-trigonoliimines A and B (class
A) and the R-configuration at the stereogenic center of
(−)-trigonoliimine C (class B),7 suggesting that these two
classes of trigonoliimines stem from enantiomeric hydrox-
yindolenine precursors. This provided further impetus to
develop an enantioselective synthesis of these alkaloids as we
could begin to address these questions by accessing synthetic
samples of enantiomerically enriched trigonoliimines A−C.
Synthesis of ( )-19-Desmethoxytrigonoliimine C. In
the initial stages of our studies, in order to quickly evaluate the
plausibility of our retrosynthetic analysis of trigonoliimines, we
focused on a desmethoxytrigonoliimine model system starting
from tryptamine 16 (Scheme 2).26 Recently, Hao and Liu
reported their synthetic studies of a similar desmethoxy model
system.10 When phthalimide protected tryptamine 16 was
treated with trifluoroacetic acid (TFA), the desired dimer 17
was formed in quantitative yield.27 Heterodimer 17 could be
oxidized to homodimeric 2,2′-bistryptamine 18 in the presence
of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in tetra-
hydrofuran in 58% yield. Subsequent treatment of bistrypt-
amine 18 with Davis’ oxaziridine 19 afforded hydroxyindole-
nine ( )-20 in quantitative yield. After further attempts to
optimize this synthetic sequence, we found that treatment of
heterodimer 17 with Davis’ oxaziridine 19 (2.2 equiv) afforded
the desired hydroxyindolenine ( )-20 in 89% yield. Notably,
treatment of 17 with excess Davis’ oxaziridine 19 (4 equiv) did
Our biosynthetically inspired retrosynthesis predicted the S-
configuration on the stereogenic centers of trigonoliimines A−
C (1−3) resulting from common hydroxyindolenines 9 and 10
with S-configuration at their stereogenic centers (Scheme 1).
Interestingly, the isolation report predicted the S-configuration
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a
with the ketone moiety (vide infra), but noticed a broadening
of H NMR peaks at C11 possibly due to a rapid equilibration
Scheme 2. Synthesis of Hydroxyindolenine ( )-20
1
between imine and aminal structures. Interestingly, imine
( )-24 could also be accessed from hydroxyaminal ( )-23
28
upon heating with Sc(OTf)3 followed by treatment of the
resulting indoxyl intermediate with titanium ethoxide. When
pentacycle 24 was treated with N-formylimidazole, N24 was
formylated to give ( )-19-desmethoxy-trigonoliimine C (25)
in 75% yield. 1H NMR peaks of the formylated product ( )-25
were sharper and better resolved compared to intermediate 24,
consistent with the decreased nucleophilicity of N24.
Synthesis of the Heterodimeric 2,2′-Bistryptamine.
Promising results described above in the initial phase of this
program prompted investigation of the synthesis of hetero-
dimeric 2,2′-bistryptamine 15 (Scheme 1), which can be
derived from the union of tryptamine 16 and 6-methoxytrypt-
amine 26.27b,29 Initial heterodimerization attempts with trypt-
amine 16 and 6-methoxytryptamine 26 under ionic conditions
yielded dimeric tryptamine 17 as a single diastereomer in 22%
yield (Scheme 4). However, no desired heterodimer could be
a
Conditions: (a) TFA, 23 °C; (b) DDQ, THF, 23 °C; (c) 19 (1.1
equiv), CH2Cl2, 23 °C; (d) 19 (2.2 equiv), CH2Cl2, 23 °C.
not result in the formation of overoxidized products consistent
with our original hypothesis.17
With ready access to hydroxyindolenine ( )-20, we
investigated the Wagner−Meerwein type 1,2-alkyl shift25 to
form indoxyl ( )-21, which possesses the structural backbone
of trigonoliimine C (3, Scheme 1). When hydroxyindolenine
( )-20 was treated with scandium trifluoromethanesulfonate
[Sc(OTf)3] in toluene at 85 °C,18 the desired indoxyl 21 was
obtained in 36% yield along with oxindole 22 (32% yield,
Scheme 3) as a byproduct. Interestingly, treatment of
Scheme 4. Attempted Heterodimerization of Tryptamines 16
a
and 26 under Ionic Conditions
Scheme 3. Synthesis of ( )-19-Desmethoxytrigonoliimine C
a
(25)
a
Conditions: (a) TFA, 23 °C.
obtained in any synthetically useful amount. Hence, for the
selective union of two tryptamine subunits, we envisioned the
use of a metal-catalyzed and directed cross-coupling reaction.
The union of two indole subunits at their 2,2′-junction by
virtue of a transition-metal-catalyzed cross-coupling reaction is
well-precedented.30,31 However, to the best of our knowledge,
there had been no reports that cross-couple two N1-
unprotected indole subunits. Hence, we focused our attention
on direct assembly of two N1-unprotected tryptamine subunits
by application of the Suzuki−Miyaura cross-coupling reac-
tion.32
For the synthesis of the boronate coupling partner, we
planned to achieve a direct C2 borylation of 6-methoxytrypt-
amine 26 by using an iridium-based catalyst system developed
by Ishiyama, Miyaura, and Hartwig (Table 1).33 Initial attempts
with pinacolborane (1 equiv) in the presence of 5 mol % of
iridium catalyst and 10 mol % of bispyridine ligand in
tetrahydrofuran at 70 °C did not afford any desired product
(Table 1, entry 1). When 6-methoxytryptamine 26 was treated
with pinacolborane (2.6 equiv) at 80 °C, only diborylated
product 28 was obtained in 93% yield (Table 1, entry 2). After
optimization (Table 1, entries 2−5), we found that 6-
methoxytryptamine 26 could be converted selectively to
monoborylated tryptamine 27 in 67% yield by exposure to
pinacolborane (2.2 equiv), 10 mol % of iridium catalyst, and 10
mol % of bispyridine ligand in dichloromethane at 23 °C
(Table 1, entry 6).
a
Conditions: (a) Sc(OTf)3, PhMe, 85 °C; (b) NH2NH2·H2O, MeOH,
80 °C; (c) Ti(OEt)4, THF, 40 °C; (d) Sc(OTf)3, MeCN, PhMe (2:1),
81 °C;28 (e) N-formylimidazole, THF, 23 °C.
hydroxyindolenine 20 with 20 equiv of hydrazine in refluxing
methanol afforded the cyclized hydroxyaminal 23 as a major
isomer in 99% yield. While the H NMR analysis of aminal 23
in deuterated chloroform was consistent with the structure
shown in Scheme 3, H NMR analysis of the same sample in
deuterated methanol revealed the presence of multiple species
consistent with structural flexibility of this intermediate (vide
infra).
With synthetic access to indoxyl ( )-21, we were set to
answer the question of whether the intramolecular condensa-
tion would occur between N12−C15 or N24−C15. Hydrazi-
nolysis of indoxyl ( )-21 and subsequent treatment of the
resulting crude mixture with titanium tetraethoxide yielded
seven-membered imine ( )-24 in 49% yield over two steps
(vide infra). Notably, we did not observe the formation of the
5-membered imine resulting from the condensation of N24
1
1
We next sought conditions that enabled the efficient union of
two tryptamine subunits to form 2,2′-bistryptamine 32. Initial
cross-coupling attempts using various palladium sources in
conjunction with XPhos34 and potassium phosphate in
toluene−water mixture at 110 °C provided the desired product
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Table 1. Optimization of the Iridium-Catalyzed Borylation at
C2 of 6-Methoxytryptamine 26
Table 2. Optimization of the Suzuki−Miyaura Cross-
Coupling Reaction for the Formation of 2,2′-Bistryptamine
32
bipyridyl
b
HBPin Ir catalyst
ligand
temp
entry halide
base
solvent
yield (%)
a
entry (equiv) (mol %)
(mol %)
(°C)
solvent
result
1
2
3
4
5
6
7
8
9
29
29
29
30
30
30
30
30
30
29
30
K3PO4
KOH
PhMe, H2O (5:1)
PhMe, H2O (5:1)
THF, H2O (5:1)
PhMe, H2O (5:1)
PhMe, H2O (5:1)
PhMe, H2O (5:1)
PhMe, H2O (5:1)
PhMe, H2O (5:1)
PhMe, H2O (5:1)
PhMe, H2O (5:1)
PhMe, H2O (5:1)
40
30
8
1
2
3
1
5
3
5
10
6
70
80
45
THF
THF
THF
no reaction
2.6
2
28: 93%
Tl2CO3
10
27:28 =
K3PO4
31
19
46
60
33
63
64
56
b
2.2:1 ,
K2CO3
48%
c
K3PO4 + Ag(OTf)
K3PO4 + Ag2CO3
Ag2CO3
4
5
2
5
5
10
10
45
45
CH2Cl2 27: 55%
d
2.5
CH2Cl2 27:28 =
b
4.5:1 ,
66%
Ag3PO4
6
2.2
10
10
23
CH2Cl2 27: 67%
10
Ag3PO4
a
b
1
e
Isolated yield. Ratio of 27 to 28 was determined by H NMR.
11
Ag3PO4
a
Uniform conditions unless otherwise noted: precatalyst 31 (20 mol
%), base (2.0 equiv), halide (1 equiv), boronate (1.2 equiv), 23 °C.
32, albeit in low and variable yields (7−44%). Hence, we
sought to perform this carbon−carbon bond formation under
milder conditions. When bromide 29 and boronic ester 27 were
treated with the Buchwald biphenyl precatalyst 31 and
potassium phosphate in toluene−water mixture at 23 °C,35
the desired bistryptamine heterodimer 32 was formed in 40%
yield (Table 2, entry 1). We discovered that the addition of a
halophilic silver salt to the reaction mixture induced a notable
increase in product yield (Table 2, entries 6 and 7).36 Notably,
the use of Ag2CO3 (2.0 equiv) as the sole base resulted in a
lower yield (33%, Table 2, entry 8) of the desired product 32.
When we used Ag3PO4 (2.0 equiv) as the sole base additive, we
were able to obtain the desired product in 63% yield (Table 2,
entry 9), possibly due to the 2-fold role of this base as both a
halophile and ideal base additive.
Oxidation of Bistryptamine 32. Unlike the oxidation of
homodimeric bistryptamine 18 used in our model system
(Scheme 2), 32 consists of two indole rings with different
electronic properties, raising an interesting question regarding
the regioselectivity of the following oxidation event. When
bistryptamine 32 was treated with Davis’ oxaziridine 19 at 0 °C,
we obtained hydroxyindolenines ( )-36 and ( )-38 (36:38 =
1:1) in 91% combined yield (Table 3, entry 1). Interestingly,
exposure of neat bistryptamine 32 to air for 12 d resulted in the
formation of hydroxyindolenines ( )-36 and ( )-38 (36:38 =
1.5:1) in 27% combined yield along with 65% of unreacted
starting material 32 (Table 3, entry 2). Notably, the presence of
regioisomeric pairs is commonly observed in the trigonostemon
alkaloids family (Figure 1).37 Hence, we reasoned that the
formation of both regioisomers during the oxidation of
bistryptamine precursor might explain the biodiversity observed
in the trigonostemon alkaloids family. Notably, modification of
bistryptamine 32 through an N1-protection with various
electron-withdrawing groups greatly altered their chemical
properties toward oxidation. Phenylsulfonyl, methylsulfonyl,
and tert-butoxycarbonyl protection of N1 of 32 completely
suppressed their oxidation under the conditions previously
b
c
d
Isolated yield (%). K3PO4 (2.0 equiv), Ag(OTf) (6.0 equiv). K3PO4
e
(2.0 equiv), Ag2CO3 (1.0 equiv). Precatalyst 31 (10 mol %).
described using Davis’ oxaziridine 19 (Table 3, entries 3, 4, and
6). When substrate 35 was treated with DMDO, the desired
hydroxyindolenine could be formed as a single regioisomer,
albeit in only 32% yield (Table 3, entry 7). However, substrate
35 was found to be inert under representative asymmetric
oxidation conditions developed by Shi38 and Sharpless39 (Table
3, entries 8 and 9).
When unprotected bistryptamine 32 was oxidized under
Sharpless39 or Miller23 asymmetric oxidation conditions,
hydroxyindolenines (+)-36 and (+)-38 could be obtained
albeit in low enantiomeric excess (Table 3, entries 10 and 11).
Gratifyingly, we discovered that (+)-((8,8-dichlorocamphoryl)-
sulfonyl) oxaziridine 41, originally reported by the Davis
research group,40 afforded hydroxyindolenines (+)-36 and
(+)-38 (36:38 = 2.2:1) in 95% yield and with an outstanding
level of enantioselection for both regioisomers (96% ee, Table
3, entry 12).8 We envisioned that hydroxyindolenine (+)-36
would serve as a synthetic precursor for trigonoliimines A (1)
and C (3) and hydroxyindolenine (+)-38 as a precursor for
trigonoliimine B (2) and isotrigonoliimine C (4).
Total Synthesis of (−)-Trigonoliimines A and B. We
next aimed to synthesize (−)-trigonoliimines A (1) and B(2)
from hydroxyindolenines (+)-36 and (+)-38. Consistent with
our model studies (Scheme 3), hydrazinolysis of hydroxyindo-
lenines (+)-36 and (+)-38 afforded the cis-fused aminals (+)-7
and (+)-8 (7:8 = 2.2:1) in 99% combined yield through
intramolecular cyclizations (Scheme 5). Aminals (+)-7 and
(+)-8 were separable at this stage to enable their chemical
examination independently. However, during scale up they
were easily processed together and separated at a more practical
stage in the synthesis (vide infra, Scheme 7).
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Table 3. Oxidation of 2,2′-Bistryptamine Derivatives
entry
substrate
condition
yield (product)
% ee (product)
a
1
32
32
33
34
34
35
35
35
35
32
32
32
19, CH2Cl2, 0 °C
91% (36:38 = 1:1)
27% (36:38 = 1.5:1)
no reaction
a,b
2
air, neat, 23 °C, 12 d
19, CH2Cl2, 23→50 °C
19, CH2Cl2, 23 °C
m-CPBA, CH2Cl2, 23 °C, 3 h
19, CH2CI2, 23→65 °C
3
4
no reaction
5
decomposition
no reaction
6
7
DMDO, acetone, 23 °C, 11 h
32% (37)
c
8
Shi epoxidation
no reaction
d
d
9
Sharpless asymmetric dihydroxylation
Sharpless asymmetric dihydroxylation
no reaction
a
a
a
10
11
12
39% (36:38 = 1.7:1)
49% (36:38 = 1.3:1)
<3 (36), <3 (38)
20 (36), <5 (38)
96 (36), 96 (38)
40 (10 mol %), DMAP, H2O2, DIC, CHCl3, 0 °C, 24.5 h
41, CH2Cl2, 30→23 °C
95% (36:38 = 2.2:1)
a
b
c
1
The ratio of 36 to 38 was determined by H NMR. 65% of unreacted starting material 33 was isolated. Shi catalyst (39), Na2B2O7·H2O, n-
d
Bu4NHSO4, oxone, K2CO3, EDTA, H2O, DMM, MeCN. AD-mix-α, methanesulfonamide, t-BuOH, H2O.
a
Scheme 5. Formation and Attempted Stereoselective Rearrangement of Hydroxyaminals (+)-7 and (+)-8
a
Conditions: (a) NH2NH2·H2O, MeOH, 80 °C, 99%; (b) TFE, 105 °C.
When aminal (+)-7 (96% ee) was heated at 105 °C in
trifluoroethanol (TFE), rearranged azepane 5 was obtained in
34% yield, albeit with significantly eroded enantiomeric excess
(15% ee). On the other hand, when aminal (+)-8 (96% ee) was
subjected to identical reaction conditions, only decomposition
of the starting material was observed. In the latter case, we
reasoned that the absence of the electron donating methoxy
group at C16′ results in less efficient ionization of hydroxyl
group at C20′. While the NMR analysis of aminals (+)-7 and
(+)-8 in deuterated chloroform was consistent with cis-fused
pentacyclic structures, analysis of the same synthetic samples in
deuterated methanol showed the presence of multiple species,
consistent with formation of aminal and imine isomers
(Scheme 6). We rationalized that the erosion of ee was due
to a low level of diastereoselection during the N12−C24 bond
formation followed by reversible ionization of C20 in the form
of intermediate 44. Alternatively, the C20 dehydroxylation of
intermediate 42, or the corresponding solvent adduct, would
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Scheme 6. Plausible Mechanism for the Erosion of Enantiomeric Excess during the Formation of 5
result in overall N12−C20 bond formation with low level of
stereochemical control (Scheme 6).
aminals (+)-7 and (+)-8 upon treatment with Martin sulfurane
to give iminium ions 48 and 49. The fused bicyclic structure of
intermediates 48 and 49 allows N12 to attack only from the α-
face, resulting in a stereoretentive cyclization. The expulsion of
the aniline nitrogen from aminal intermediates 50 and 51
would result in the formation of azepanes (−)-5 and (−)-6. X-
ray crystal structure analysis of pentacycle (−)-6 (Scheme 7),
the direct precursor of (−)-trigonoliimine B (2), unambigu-
ously confirmed the molecular structure and assigned the S-
configuration at C20, corroborating the stereoretentive mode of
N12−C20 cyclization.
Based on these observations, we reasoned that the activation
of the hydroxyl groups of aminals (+)-7 and (+)-8 under mild
conditions in an aprotic solvent would be ideal in order for this
transformation to proceed with minimal erosion of enantio-
meric excess. Hence, we treated a solution of aminals (+)-7 and
(+)-8 (7:8 = 2.2:1) in dichloromethane with Martin sulfurane41
at −78 °C, which provided the desired azepanes (−)-5 and
(−)-6 in 47% combined yield (28% and 19% yield, respectively,
Scheme 7). Azepanes (−)-5 and (−)-6 could be readily
separated using flash column chromatography at this stage.
Importantly, azepanes (−)-5 and (−)-6 were obtained with
minimal erosion of enantiomeric excess (94% ee and 95% ee,
respectively). A plausible mechanism for this transformation
involves an activation of hydroxyl moiety of the cis-fused
For the completion of the total synthesis of (−)-trigonolii-
mine A (1), we initially treated the azepane (−)-5 with a
catalytic amount of pyridinium p-toluenesulfonate (PPTS) in
trimethylorthoformate at 50 °C (Table 4, entry 1).42 Under
Table 4. Completion of the Total Synthesis of
(−)-Trigonoliimine A (1)
Scheme 7. Stereoselective Formation of Azepanes (−)-5 and
(−)-6
PPTS
(equiv)
temp
(°C)
1
(%)
52
(%)
entry
orthoformate + solvent
1
2
3
0.1
2
CH(OMe)3 (solvent)
50
45
23
16
49
82
61
37
15
CH(O-i-Pr)3, CH2Cl2 (1:1)
3
CH(O-i-Pr)3 (10 equiv),
CH2Cl2
these conditions, we were able to obtain the first synthetic
sample of (−)-trigonoliimine A (1), albeit in low yield (16%).
A possible pathway for the formation of (−)-trigonoliimine A
(1) (path A) and byproduct (+)-52 (path B) from azepane
(−)-5 is shown in Scheme 8. Conversion of (−)-5 to (−)-1
involves imidate formation at N13 followed by cyclization with
N12.43 Interestingly, the major product formed under these
conditions was hexacyclic imine 52 (61%, Scheme 8). Initial
reaction of N23 of azepane (−)-5 with the oxocarbenium ion
generated from trimethylorthoformate may form the iminium
ion 56 (path B), and the resulting electrophilic C24 would be
trapped with N13 to form intermediate 57. The N23 released
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a
Scheme 8. Plausible Mechanism for the Formation of
(−)-Trigonoliimine A (1) and Byproduct (+)-52
Scheme 9. Total Synthesis of (−)-Trigonoliimine B (2)
a
Conditions: (a) CH(O-i-Pr)3, PPTS, CH2Cl2, 23 °C.
(−)-trigonoliimine C (3) and (−)-isotrigonoliimine C (4)
from the same versatile hydroxyindolenines (+)-36 and (+)-38
described above via a divergent synthetic path employing a
Wagner−Meerwein-type 1,2-alkyl rearrangement.25 Literature
precedent and prior synthetic studies from our laboratory
suggested that hydroxyindolenines derived from the oxidation
of 2-aryl-3-alkyl-disubstituted indoles can rearrange to the
corresponding indoxyls, oxindoles, or mixtures of both
depending on the reaction conditions.19,44 Hence, we turned
our attention to finding conditions that would favor the
selective formation of indoxyls (−)-60 and (−)-61 over the
oxindole byproducts (Table 5). Initially, we examined various
lanthanide trifluoromethanesulfonates as additives for the
rearrangement of hydroxyindolenines (+)-36 and (+)-38
Table 5. Rearrangement of Hydroxyindolenines (+)-36 and
(+)-38 in the Presence of Various Lewis Acids
% yield of
temp time
indoxyls
a
entry
L.A. (equiv)
solvent
MeCN
(°C)
(h)
(60:61)
from the aminal moiety of 57 will give the pentacyclic
intermediate 58. Intramolecular cyclization resulting from the
N12 attack to the imidate moiety of 58 followed by methanol
elimination would yield the isomeric hexacyclic byproduct
(+)-52.
1
2
3
4
5
6
7
8
9
Sc(OTf)3 (3)
Dy(OTf)3 (3)
La(OTf)3 (3)
Pr(OTf)3 (3)
Ho(OTf)3 (3)
Eu(OTf)3 (3)
Nd(OTf)3 (3)
Gd(OTf)3 (3)
Eu(OTf)3 (3)
65
65
65
65
65
65
65
65
65
45
45
39
14
39
14
14
14
24
12 (60 only)
35 (1.7:1)
50 (1.6:1)
42 (2:1)
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
44 (2:1)
In order to suppress the undesired reactivity at N23 of (−)-5,
we examined its pretreatment with PPTS (2 equiv) followed by
exposure to triisopropyl orthoformate, a more sterically
crowded reagent. As expected, the desired (−)-trigonoliimine
A (1) was obtained as a major product (49%, Table 4, entry 2).
After further optimization, we found that pretreatment of
azepane (−)-5 with PPTS (3 equiv) in dichloromethane at 23
°C, followed by addition of triisopropylorthoformate (10
equiv) afforded the desired (−)-trigonoliimine A (1) in 82%
50 (1.3:1)
41 (1.4:1)
45 (1.4:1)
46 (1.3:1)
PhMe, MeCN
(1:1)
10
11
12
13
14
15
16
17
18
19
20
Eu(OTF)3 (3)
La(OTf)3 (3)
La(OTf)3 (3)
La(OTf)3 (3)
La(OTf)3 (1)
La(OTf)3 (0.5)
La(OTf)3 (0.5)
La(OTf)3 (0.5)
Eu(OTf)3 (0.5)
La(OTf)3 (0.5)
Eu(OTf)3 (0.5)
MeOH
65
90
65
65
65
65
80
23
65
65
72
45
14
55
19
19
15
19
72
18
42
38
no reaction
40 (1:1.2)
49 (1.3:1)
49 (1:1.2)
57 (1:1.1)
52 (1:1.1)
56 (1:1.2)
no reaction
42 (1.5:1)
52 (1.3:1)
48 (1.2:1)
PhMe
THF
ClCH2CH2Cl
ClCH2CH2Cl
ClCH2CH2Cl
PhMe
yield [[α]24 = −294 (c 0.24, CHCl3)] (Table 4, entry 3).
D
Under identical reaction conditions, azepane (−)-6 was
converted to (−)-trigonoliimine B (2) in 94% yield [[α]24
=
D
−352 (c 0.32, CHCl3), Scheme 9]. All H and 13C NMR data
for our synthetic (−)-trigonoliimines A (1) and B (2) matched
those provided in the isolation report,7 confirming the
molecular structure of these alkaloids.
1
ClCH2CH2Cl
MeCN
MeCN
MeCN
Total Synthesis of (−)-Trigonoliimine C and (−)-Iso-
trigonoliimine C. We also wished to gain access to
a
1
The ratio of 60 to 61 was determined by H NMR.
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(36:38 = 2.2:1, Table 5). In general, we were able to obtain
indoxyls (−)-60 and (−)-61 in moderate yield (Table 5, entries
3−9, 11−16, 18−20). The use of methanol as solvent did not
provide the desired indoxyl products in the presence of
europium trifluoromethanesulfonate at 65 °C (Table 5, entry
10). We observed that the choice of solvent had a significant
effect in products ratio. When acetonitrile, acetonitrile−toluene
mixture, or tetrahydrofuran was used as solvent indoxyl (−)-60
was obtained as a major product. On the other hand, when
toluene or 1,2-dichloroethane was used as solvent indoxyl
(−)-61 was formed in slight excess (Table 5, entries 11, 13−
16). The major byproduct observed in these reactions was
oxindole (+)-62 (34% for Table 5, entry 16). Oxindole
byproduct (+)-62 did not show any reactivity upon treatment
with three equivalents of lanthanum trifluoromethanesulfonate
in 1,2-dichloroethane at 65 °C. Similarly, when the indoxyl
(−)-60 was treated with three equivalents of lanthanum
trifluoromethanesulfonate in 1,2-dichloroethane at 65 °C, no
reactivity was observed.
Scheme 11. Selective Rearrangement of Hydroxyindolenine
63 to Indoxyl 64
a
a
Conditions: (a) TFE, 105 °C.
Based on these observations, we heated hydroxyindolenines
(+)-36 and (+)-38 (36:38 = 2.2:1) in TFE at 102 °C and
obtained indoxyls (−)-60 and (−)-61 (60:61 = 2.2:1)
selectively in 93% yield without any formation of the undesired
oxindole (Scheme 12). X-ray crystal structure analysis of the
enantiomerically enriched (96% ee) C18-bromide derivative of
(−)-60 enabled us to unequivocally assign the S-configuration
at C14.8
Due to the nontrivial separation of regioisomeric hydrox-
yindolenines (+)-36 and (+)-38 by flash column chromatog-
raphy, prep-HPLC enabled us to access pure samples of both
hydroxyindolenines (+)-36 and (+)-38 for their separate
characterization and evaluation of chemical properties. When
hydroxyindolenine (+)-36 was treated with three equivalents of
europium trifluoromethanesulfonate in acetonitrile at 72 °C,
indoxyls (−)-60 and (−)-61 (48%, 60:61 = 4.5:1) and oxindole
(+)-62 (50%) were obtained (Scheme 10).45 On the other
Scheme 12. Selective 1,2-Alkyl Shift of Hydroxyindolenines
(+)-36 and (+)-38
a
Scheme 10. Eu(OTf)3-Mediated Rearrangement of
(+)-Hydroxyindolenines (+)-36 and (+)-38
a
a
Conditions: (a) TFE, 102 °C.
The synthetic utility of asymmetric oxidation followed by
stereospecific rearrangement of 2-aryl-3-alkyl-indole derivatives
holds great promise for other applications as illustrated in
Scheme 13. The 1,2-alkyl shift of hydroxyindolenine (+)-65,
obtained through Miller’s asymmetric oxidation of the
corresponding 2-aryltryptamine,23 gave indoxyl (+)-66, which
constitutes the core structure of hinckdentine A (67).46
Similarly, when tryptophol derivative 68 was subjected to
Miller’s asymmetric oxidation23 followed by base mediated 1,2-
Scheme 13. Further Synthetic Application of Asymmetric
Oxidation and 1,2-Alkyl Shift of 2,3-Disubstituted Indole
Derivatives
a
a
Conditions: (a) Eu(OTf)3, MeCN, 72 °C.
hand, upon treatment with three equivalents of europium
trifluoromethanesulfonate in acetonitrile at 72 °C, hydrox-
yindolenine (+)-38 afforded indoxyl (−)-61 as the sole product
in 62% yield (Scheme 10).
While most of the reaction conditions that utilized lanthanide
Lewis acids produced oxindoles as byproducts during the
rearrangement of hydroxyindolenines (+)-36 and (+)-38,
promising results were obtained during our study directed
toward the total synthesis of (−)-trigonoliimine A (1). Inspired
by the transformation of aminal (+)-7 to azepane 5 upon
heating in TFE (Scheme 5), we attempted to directly access
trigonoliimine A (1) by exposing formylated hydroxyindolenine
63 under the same reaction conditions. Interestingly, when the
formylated hydroxyindolenine 63 was heated in TFE at 105 °C,
indoxyl 64 was selectively formed in 59% yield (Scheme 11).
a
Conditions: (a) HFIP, HCOOH (10:1), 90 °C; (b) 40, DMAP,
H2O2, DIC, CHCl3, 0 °C; (c) t-BuOK, t-BuOH, 23 °C.
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alkyl shift sequence, indoxyl (−)-70 was obtained in 79% ee,
providing a formal synthesis of (−)-hinckdentine A (67,
Scheme 13).42 Notably, the indoxyl 70 in racemic form has
served as a key intermediate in Kawasaki’s total synthesis of
( )-hinckdentine A (67).42
With robust access to indoxyls (−)-60 and (−)-61 secured,
we next investigated their hydrazinolysis. When indoxyl (−)-60
was treated with 10 equiv of hydrazine hydrate in methanol at
80 °C, indoxyl 71 was obtained in 81% yield (Scheme 14).
a
Scheme 14. Hydrazinolysis of Indoxyls (−)-60 and (−)-61
Figure 2. Calculated equilibrium geometries of 7-membered imine 11
and 5-membered imine 73.
energy of 7-membered imine 11 and 5-membered imine 73
predicted that imine 11 is 9.4 kcal/mol lower in energy (Figure
2).47
a
Conditions: (a) NH2NH2·H2O, MeOH, 90 °C.
Consistent with the model studies (Scheme 3), treatment of
However, when indoxyl (−)-61 was exposed to the same
reaction conditions, lower and variable yields (8−45%) of the
indoxyl 72 were obtained (Scheme 14). We reasoned that the
absence of the methoxy group at C19 of indoxyl 72 rendered
the carbonyl group more electrophilic, which resulted in the
formation of polar hydrazone byproducts.
imines (−)-11 and (−)-12 (11:12 = 2:1) with freshly prepared
24
N-formylimidazole afforded (−)-trigonoliimine C (3) [[α]D
=
−147 (c 0.12, CHCl3)] and (−)-isotrigonoliimine C (4)
[[α]24 = −220 (c 0.10, CHCl3)] in 57% and 16% yield,
D
respectively (Scheme 16). All H and 13C NMR data of our
1
Hence, in order to achieve higher efficiency and practicality,
we envisioned performing the hydrazynolysis of indoxyls
(−)-60 and (−)-61 and the condensation of the resulting
intermediates in a one-pot, two-step procedure. We predicted
that indoxyls (−)-71, (−)-72, and hydrazone byproducts
formed during the hydrazinolysis of indoxyls (−)-60 and
(−)-61 would be viable substrates for the subsequent titanium
ethoxide-mediated condensation reaction. Indeed, treatment of
indoxyls (−)-60 and (−)-61 (60:61 = 2.2:1) with 10 equiv of
hydrazine hydrate in methanol at 80 °C followed by
concentration of the reaction mixture and addition of the
tetrahydrofuran solution of titanium ethoxide and heating at 42
°C afforded the desired pentacyclic imines (−)-11 and (−)-12
(11:12 = 2:1) in 61% yield over two steps (Scheme 15).
Scheme 16. Total Synthesis of (−)-Trigonoliimine C (3) and
a
(−)-Isotrigonoliimine C (4)
a
Conditions: (a) N-formylimidazole, THF, 23 °C.
synthetic (−)-trigonoliimines C (3) matched those provided in
the isolation report.1 While regioisomeric intermediates
mentioned above could be separated earlier in the synthetic
sequence for independent examination,8 the ease of separation
of (−)-trigonoliimine C (3) and (−)-isotrigonoliimine C (4)
by silica gel column chromatography allowed a practical
processing of the earlier intermediates as mixtures until the
final stage of the synthesis.48
Scheme 15. One-Pot, Two-Step Synthesis of Imines (−)-11
a
and (−)-12
The sign and magnitude of specific rotation of our synthetic
trigonoliimines in conjunction with our X-ray crystal structure
data provided valuable information regarding the stereo-
chemistry of these alkaloids. First, our synthetic trigonoliimines
A−C (1−3) derived from hydroxyindolenines (+)-36 and
(+)-38 exhibited negative signs in their specific rotation (Table
6). However, naturally occurring trigonoliimines A (1) and B
(2) were reported to have a positive sign in their specific
rotations whereas trigonoliimine C (3) was reported to have a
negative sign in its specific rotation. These observations, in
conjunction with our X-ray crystal structures, which unambig-
uously corroborated the absolute stereochemistry of our
synthetic (−)-trigonoliimines A−C (1−3), enabled the revision
of the absolute stereochemistry of all trigonoliimines (Figure
a
Conditions: (a) NH2NH2·H2O, MeOH, 80 °C; (b) Ti(OEt)4, THF,
42 °C.
Consistent with our earlier observations in the model system
(Scheme 3) and the molecular structure of the natural product
trigonoliimine C (3), we did not observe the formation of the
5-membered imine resulting from the condensative cyclization
of N24 onto the ketone moiety (Figure 2). Notably, DFT
calculation (B3LYP, 6.31G*) of the equilibrium geometries and
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Table 6. Specific Rotation Values of Natural and Synthetic
Trigonoliimines A−C (1−3)
Table 7. Anticancer Activity of All Known Trigonoliimines
and Structurally Related Alkaloids
a
a
entry
1
alkaloids
natural ([α]10
)
nynthetic ([α]10
)
D
entry
compd
HeLa (μM)
U-937 (μM)
44
D
trigonoliimine A
+13.3 (c 0.3, CHCl3)
+5.0 (c 0.5, CHCl3)
−4.8 (c 0.45, CHCl3)
−294 (c 0.24, CHCl3,
1
(−)-1
( )-1
(−)-2
(−)-3
( )-3
(−)-4
(−)-11·CD3CO2D
(−)-12·CD3CO2D
( )-23
>50
9
94% ee)
2
>50
21.3 4.5
34.6 7.1
20.3 3.1
29.6 9.5
>35
2
3
trigonoliimine B
trigonoliimine C
−352 (c 0.32, CHCl3,
3
>50
95% ee)
4
>50
−147 (c 0.12, CHCl3,
5
>100
96% ee)
6
>50
a
% ee of the key precursor for the corresponding synthetic
trigonoliimine.
7
29.7 3.1
>100
10.9 0.2
38.6 9.5
19.2 4.4
10.3 3.0
0.73 0.05
35.6 9.2
7.9 0.5
22.8 2.2
>50
8
9
>50
10
11
12
13
14
15
( )-24
>30
1). Additionally, all of our synthetic (−)-trigonoliimines A−C
(1−3) showed a significantly larger magnitude of specific
rotations compared to those reported for the naturally isolated
samples (Table 6). Importantly, the enantiomeric excess of our
samples has been quantified by HPLC analysis of enantiomeri-
cally enriched samples of several intermediates against readily
available racemic samples from our exploratory studies in this
program. A potential explanation for the smaller magnitude of
the specific rotations of the naturally occurring trigonoliimines
is their possible natural formation via partial resolution after a
nonselective oxidation of a precursor such as bistryptamine 15
(Scheme 1). Our observations regarding the propensity of
bistryptamine 32 to undergo air oxidation (Table 3, entry 2)
lends further credence to this hypothesis.8,49
Testing of all known Trigonoliimines and Structurally
Related Alkaloids for Their Anticancer Activity. Despite
the numerous synthetic efforts targeting the trigonoliimine class
of natural products, only one study has investigated their
biological activity.7 This report identified modest anti-HIV
activity for trigonoliimine A (0.95 μg/mL, 2.7 μM). Inspired by
the potent anticancer activities of indolocarbazole glycopeptides
with 2,2′-connectivity,2 we set out to test the anticancer
activities of trigonoliimines A−C and various synthetic
intermediates generated from this synthetic program.
Our synthetic work has enabled a study of the anticancer
activities of all known trigonoliimines as well as numerous
advanced intermediates, many of which share similar cores to
the natural products. Thirteen compounds were identified as
preliminary hits for their ability to induce cell death and were
carried on for further testing in two cancer cell lines (U-937,
human lymphoma, and HeLa, human cervical cancer). The 72-
h IC50 values in both U-937 and HeLa cells for the lead
compounds is shown in Table 7. In general, most of the
compounds, including trigonoliimines A−C, showed weak
activity (10−50 μM) in both cell lines.
TFA-d1 salt of amine ( )-24, a precursor to trigonoliimine C
(3), showed excellent activity in U-937 cells (730 nM).
However, when comparing it to free amine of ( )-24 and
acetic acid-d4 salt of (−)-11 and (−)-12, which are all
structurally related compounds, the TFA-d1 salt of ( )-24
was much more active than would be predicted from their
subtle structural differences. As TFA is not able to induce
cancer cell death on its own, this suggests that the activity of the
TFA salt of amine ( )-24 arises from a specific interaction
resulting from the TFA salt. This is consistent with previous
reports that TFA and TFA salts affect cell proliferation and
protein synthesis in cells.50 Tryptophol derivative 68 was also
reasonably active against both U-937 and HeLa cells. However,
68 contains a nitroaromatic group which is known to behave
promiscuously in cells.51 Hence, further biological studies
( )-24·CF3CO2D
(+)-52
8.6 1.8
>100
68
3.6 1.1
13.6 6.3
35.8 8.6
( )-69
b
(+)-69
a
72 h IC50 values in HeLa (human cervical cancer) or U-937 (human
lymphoma) cell lines as monitored by SRB (HeLa) or MTS (U-937).
Error is standard deviation of the mean, n = 3. SRB: sulforhodamine B.
MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
b
sulfophenyl)-2H-tetrazolium. 43% ee.
would be necessary to unequivocally address anticancer
activities of these compounds.
CONCLUSION
■
We have described the development of our synthetic strategy
for the enantioselective total synthesis of all known
(−)-trigonoliimines A (1), B (2), and C (3). Rapid access to
the key hydroxyindolenines (+)-36 and (+)-38 was enabled by
a Suzuki−Miyaura cross-coupling reaction using Buchwald’s
precatalyst 31 in conjunction with silver phosphate at ambient
temperature, followed by a highly enantioselective oxidation at
the enantiodetermining branching point of our syntheses.
Stereoretentive and condensative cyclization of cis-fused
aminals (+)-7 and (+)-8, obtained from the phthalimide
deprotection of (+)-36 and (+)-38, gave azepanes (−)-5 and
(−)-6 as the direct precursors of (−)-trigonoliimines A (1) and
B (2). Hence, the total synthesis of (−)-trigonoliimines A (1)
and B (2) were achieved in seven steps from commercially
available material. Our succinct enantioselective syntheses of
(−)-trigonoliimines C (3) and (−)-isotrigonoliimine C (4) are
each eight steps from commercially available material and draw
on a selective Wagner−Meerwein-type 1,2-alkyl shift of
hydroxyindolenines (+)-36 and (+)-38, which gave indoxyls
(−)-60 and (−)-61.
Our enantioselective approach to these natural products
allowed the revision of the absolute stereochemistry of alkaloids
(−)-1−3 and raised interesting questions related to the
apparent low enantiomeric excess of the naturally occurring
trigonoliimines. Furthermore, our synthetic efforts have
enabled the testing of these alkaloids and several advanced
intermediates in two cancer cell lines, revealing mild activity for
certain members of this class. The concise total synthesis of
(−)-trigonoliimines A−C (1−3) highlights the power of
retrobiosynthetic analysis52 as a source of inspiration in
antithetic simplification of natural product targets.
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132.4, 131.7, 130.5, 128.6, 125.4, 123.6, 123.2, 122.6, 119.8, 119.3,
118.4, 118.2, 113.1, 111.2, 109.1, 67.8, 39.0, 37.0, 33.7, 24.3. FTIR
(neat) cm−1: 3381 (br-s), 1770 (s), 1719 (s), 1616 (s), 1397 (s), 1103
(w), 751 (s). HRMS (ESI, TOF) (m/z): calcd for C36H26N4O5, [M +
H]+ 595.1976, found 595.1987. TLC (5% acetone in dichloro-
methane), Rf: 0.66 (CAM, UV).
EXPERIMENTAL SECTION
■
Experimental Procedures and Full Characterization Data for
Previously Reported Key Compounds. For complete experimental
procedures and full characterization data of all (−)-trigonoliimine
alkaloids A−C (1−3, respectively), see the Supporting Information of
our previous report of the total synthesis of all (−)-trigonoliimines.8
For complete experimental procedures and full characterization data of
the key compounds (−)-4, (−)-5, (−)-6, (+)-7, (+)-8, (−)-11,
(−)-12, 26, 27, 30, 32, (+)-36, (+)-38, (−)-60, and (−)-61 discussed
in our fully optimized route, please see the Supporting Information of
our previous report of the total synthesis of all (−)-trigonoliimines.8
(
)-2-(7,12,12b,13-Tetrahydro-6H-azepino[3,2-b:4,5-b′]-
diindol-12b-yl)ethanamine (24). Hydrazine monohydrate (33.0
μL, 0.669 mmol, 15.0 equiv) was added via syringe to a solution of
indoxyls ( )-21 (26.5 mg, 0.0446 mmol, 1 equiv) in methanol (5 mL)
under an atmosphere of argon at 23 °C, the reaction flask was
equipped with a reflux condenser, and the reaction setup was sealed
under an atmosphere of argon and heated to 80 °C. After 90 min, the
pale yellow homogeneous reaction mixture was allowed to cool to 23
°C, and the volatiles were removed under reduced pressure to afford a
pale yellow solid. A solution of titanium ethoxide (41.5 μL, 0.178
mmol, 4.00 equiv) in anhydrous tetrahydrofuran (5 mL) was added via
syringe to the yellow solid under an atmosphere of argon, and the
resulting mixture was warmed to 40 °C. After 3 h, the reaction mixture
was concentrated under reduced pressure. The crude residue adsorbed
onto silica gel (2.5 g) was dry loaded and purified by flash column
chromatography (silica gel: diameter 2 cm, ht. 10 cm; eluent: 23%
chloroform, 5.1% methanol, 0.6% ammonium hydroxide in dichloro-
methane) to afford imine ( )-24 (6.9 mg, 49%, 2 steps) as a yellow
solid. 1H NMR (500.4 MHz, CD3OD, 21 °C):53 δ 7.83 (ddd, J = 8.3,
1.2, 0.8 Hz, 1H), 7.71 (ddd, J = 8.6, 7.0, 1.2 Hz, 1H), 7.51 (td, J = 7.9,
1.0 Hz, 1H), 7.42 (td, J = 8.2, 0.9 Hz, 1H), 7.21 (ddd, J = 8.2, 7.0, 1.2
Hz, 1H) 7.11 (td, J = 8.6, 0.8 Hz, 1H), 7.08 (ddd, J = 8.0, 7.0, 1.0 Hz,
1H) 6.97 (ddd, J = 8.4, 6.9, 0.8 Hz, 1H) 4.68 (td, J = 7.9, 1.0 Hz, 1H),
4.13 (ddd, J = 14.5, 4.1, 3.1 Hz, 1H) 3.38−3.24 (m, 2H), 3.17−3.14
(m, 1H), 3.01−2.92 (m, 2H), 2.88−2.82 (m, 1H). 13C NMR (125.8
MHz, CD3OD, 21 °C):53 δ 183.2, 162.3, 143.7, 137.5, 129.4, 126.3,
124.6, 124.6, 122.4, 121.1, 119.2, 115.1, 114.5, 112.5, 111.8, 70.0, 44.9,
37.7, 36.4, 24.7. FTIR (neat) cm−1: 3272 (br-m), 1675 (s), 1636 (s),
1202 (s), 1134 (s), 723 (m). HRMS (ESI, TOF) (m/z): calcd for
C20H20N4, [M + H]+ 317.1761, found 317.1768. TLC (18% methanol,
2% ammonium hydroxide in chloroform), Rf: 0.59 (CAM, UV).
( )-19-Desmethoxytrigonoliimine C (25). Freshly prepared N-
formylimidazole54 solution (0.058 M solution in tetrahydrofuran, 0.40
mL, 23 μmol, 1.1 equiv) was added dropwise via syringe to a solution
of amine ( )-24 (6.9 mg, 22 μmol, 1 equiv) in tetrahydrofuran (1
mL) at 23 °C and placed under an argon atmosphere. After 1.5 h,
saturated aqueous sodium bicarbonate solution (4.5 mL) was added,
and the resulting mixture was diluted with dichloromethane (4.5 mL),
and the layers were separated. The aqueous layer was extracted with
dichloromethane (4.5 mL), and the combined organic layers were
dried over anhydrous sodium sulfate and concentrated under reduced
pressure. The sample of the crude residue was purified by flash column
chromatography (silica gel: diameter 2 cm, ht 10 cm; eluent: 4%
methanol, 0.4% ammonium hydroxide in chloroform) to afford
( )-19-desmethoxyltrigonoliimine C (25, 5.6 mg, 75%) as a yellow
(
)-2,2′-((3′-Hydroxy-1H,3′H-[2,2′-biindole]-3,3′-diyl)bis-
(ethane-2,1-diyl))bis(isoindoline-1,3-dione) (20). A solution of
Davis’ oxaziridine 19 (145 mg, 0.606 mmol, 2.2 equiv) in anhydrous
dichloromethane (2.5 mL) at 0 °C was transferred by cannula to a
solution of indoline derivative 17 (164 mg, 0.276 mmol, 1 equiv) in
anhydrous dichloromethane (9 mL) at 0 °C under an atmosphere of
argon. The reaction mixture was allowed to gently warm to 23 °C.
After 11 h, the reaction mixture was concentrated under reduced
pressure and the residue was purified by flash column chromatography
(silica gel: diameter 3 cm, ht 13 cm; eluent: 4% acetone in
dichloromethane) to afford hydroxyindolenine ( )-20 (121 mg,
1
73.6%) as a yellow solid. H NMR (500.4 MHz, CDCl3, 21 °C): δ
9.35 (s, 1H), 7.71 (dd, J = 5.5, 3.0 Hz, 2H), 7.61 (dd, J = 5.5, 3.0 Hz,
2H), 7.56 (dd, J = 5.6, 2.9 Hz, 2H), 7.51 (dd, J = 5.4, 3.2 Hz, 2H), 7.46
(d, J = 2.9 Hz, 1H), 7.44 (d, J = 4.0 Hz, 1H), 7.28 (d, J = 7.5 Hz, 1H),
7.11 (dt, J = 7.5, 1.2 Hz, 1H), 7.09 (d, J = 6.7 Hz, 1H), 7.02 (app-t, J =
7.5 Hz, 2H), 6.85 (ddd, J = 7.9, 7.0, 0.9 Hz, 1H), 4.08−4.02 (m, 2H),
3.64−3.58 (m, 2H), 3.54−3.48 (m, 1H), 3.46−3.39 (m, 2H), 2.68
1
(app-quintet, J = 7.1 Hz, 1H), 2.30 (app-quintet, J = 6.8 Hz, 1H). H
NMR (500.4 MHz, DMSO-d6, 21 °C): δ 10.93 (s, 1H), 7.75−7.67 (m,
6H), 7.62 (dd, J = 5.5, 3.0 Hz, 2H), 7.60 (d, J = 8.3 Hz, 1H), 7.50 (d, J
= 7.6 Hz, 2H), 7.44 (d, J = 7.6 Hz, 1H), 7.32 (dt, J = 7.6, 1.0 Hz, 1H),
7.18 (dt, J = 7.4, 0.7 Hz, 1H), 7.14 (app-t, J = 8.0 Hz, 1H), 6.92 (app-t,
J = 7.8 Hz, 1H), 6.51 (s, 1H), 4.05−3.92 (m, 2H), 3.47 (t, J = 6.8 Hz,
2H), 3.38−3.23 (m, 2H), 2.55−2.49 (m, 1H), 2.33−2.27 (m, 1H). 13C
NMR (125.8 MHz, CDCl3, 21 °C): δ 173.2, 168.7, 168.0, 154.2, 138.3,
137.0, 133.8, 133.8, 132.4, 132.0, 130.4, 128.4, 127.8, 126.1, 124.9,
123.2, 123.0, 122.6, 121.4, 120.2, 119.8, 119.2, 112.0, 86.3, 38.6, 35.4,
33.4, 24.4. 13C NMR (100.6 MHz, DMSO-d6, 21 °C): δ 174.1, 167.8,
167.2, 153.9, 139.8, 136.9, 134.0, 134.0, 131.7, 131.4, 129.3, 127.6,
127.6, 125.5, 123.6, 122.8, 122.6, 122.6, 120.4, 119.3, 118.8, 117.0,
113.0, 85.2, 37.9, 35.4, 32.8, 23.8. FTIR (neat) cm−1: 3411 (br-s),
1771 (m), 1708 (s), 1548 (m), 1397 (s), 717 (s). HRMS (ESI, TOF)
(m/z): calcd for C36H26N4O5, [M + H]+ 595.1976, found 595.1986.
TLC (5% acetone in dichloromethane), Rf: 0.4 (CAM, UV).
( )-2-(2-(2-(3-(2-(1,3-Dioxoisoindolin-2-yl)ethyl)-1H-indol-2-
yl)-3-oxoindolin-2-yl)ethyl)isoindoline-1,3-dione (21).
Scandium(III) trifluoromethanesulfonate (11.6 mg, 0.0230 mmol,
1.50 equiv) was added to a solution of hydroxyindolenine ( )-20 (9.1
mg, 15 μmol, 1 equiv) in toluene (1.5 mL) at 23 °C, and the resulting
reaction mixture was warmed to 85 °C. After 18 h, the reaction
mixture was allowed to cool to 23 °C. The reaction mixture was
diluted with dichloromethane (8 mL), saturated aqueous sodium
bicarbonate solution (8 mL) was added to the reaction mixture, and
the layers were separated. The aqueous layer was extracted with
dichloromethane (8 mL), and the combined organic layers were dried
over anhydrous sodium sulfate and concentrated under reduced
pressure. The sample of the crude residue was purified by flash column
chromatography (silica gel: diameter 1.5 cm, ht 10 cm; eluent: 1.3%
dichoromethane in acetone to 10% dichloromethane in acetone) to
afford indoxyl ( )-21 (3.3 mg, 36%) and oxindole ( )-22 (2.9 mg,
1
solid. H NMR (500.4 MHz, CDCl3, 21 °C): δ 7.94 (s, 1H), 7.59
(ddd, J = 7.8, 1.3, 0.7 Hz, 1H), 7.43 (td, J = 7.9, 1.0 Hz, 1H), 7.33−
7.30 (m, 2H), 7.10 (ddd, J = 8.2, 7.0, 1.1 Hz, 1H), 7.00 (ddd, J = 7.9,
7.0, 1.0 Hz, 1H) 6.84 (td, J = 8.1, 0.8 Hz, 1H), 6.77 (ddd, J = 7.9, 7.0,
0.8 Hz, 1H) 4.46 (ddd, J = 14.0, 11.9, 2.5 Hz, 1H) 4.09 (td, J = 12.1,
3.6 Hz, 1H), 3.29−3.23 (m, 2H) 3.16 (td, J = 16.9, 3.2 Hz, 1H) 2.75
(ddd, J = 15.3, 9.0, 5.1 Hz, 1H) 2.40 (ddd, J = 15.0, 9.1, 5.0 Hz, 1H).
13C NMR (125.8 MHz, CDCl3, 21 °C): δ 176.5, 164.0, 157.6, 137.1,
135.5, 132.2, 129.9, 124.7, 124.2, 123.1, 120.2, 120.1, 118.9, 112.5,
111.9, 110.6, 68.0, 48.5, 40.7, 35.3, 24.4. FTIR (neat) cm−1: 3285 (br-
m), 2923 (m), 1642 (s), 1611 (m), 1469 (m), 1375 (m), 1317 (m),
1141 (w), 743 (m). HRMS (ESI, TOF) (m/z): calcd for C21H21N4O
[M + H]+ 345.1710, found 345.1732. TLC (18% methanol, 2%
ammonium hydroxide in chloroform), Rf: 0.69 (CAM, UV).
1
32%) as pale yellow solids. H NMR (500.4 MHz, CDCl3, 21 °C): δ
9.08 (s, 1H), 7.87 (dd, J = 5.5, 3.0 Hz, 2H), 7.73 (dd, J = 5.5, 3.0 Hz,
2H), 7.61 (dd, J = 5.5, 2.9 Hz, 2H), 7.54−7.47 (m, 5H), 7.22 (app-t, J
= 8.9 Hz, 2H), 7.05 (ddd, J = 8.1, 7.0, 1.1 Hz, 1H), 6.97 (ddd, J = 7.9,
7.0, 1.0 Hz, 1H), 6.79 (s, 1H), 6.77 (ddd, J = 7.8, 7.1, 0.8 Hz, 1H),
3.89−3.74 (m, 4H), 3.13 (ddd, J = 10.3, 6.8, 3.1 Hz, 2H), 2.69 (app-
quintet, J = 7.4 Hz, 1H), 2.43−2.38 (m, 1H). 13C NMR (125.8 MHz,
CDCl3, 21 °C): δ 201.0, 168.8, 168.3, 161.1, 138.5, 135.5, 134.3, 134.0,
Hexacyclic Imine (+)-52. Anhydrous dichloromethane (4 mL)
was added via syringe to a flask charged with pentacycle (−)-5 (14.0
mg, 40.4 μmol, 1 equiv) and pyridinium p-toluenesulfonate (PPTS,
31.0 mg, 0.121 mmol, 3.00 equiv) at 23 °C under an atmosphere of
argon to form a bright yellow solution. After 2 min, triisopropyl
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orthoformate (93.0 μL, 0.404 mmol, 10.0 equiv) was added to the
reaction mixture. After 1 h, saturated aqueous sodium bicarbonate
solution (6 mL) was added, the resulting mixture was diluted with
dichloromethane (2 mL), and the layers were separated. The aqueous
layer was extracted with dichloromethane (2 × 6 mL), and the
combined organic layers were dried over anhydrous sodium sulfate and
concentrated under reduced pressure. The crude residue was purified
by flash column chromatography (silica gel: diameter 2 cm, ht 12 cm;
eluent: 1.8% methanol, 0.2% ammonium hydroxide in chloroform to
4.5% methanol, 0.5% ammonium hydroxide in chloroform) to afford
(−)-trigonoliimine A (1, 12 mg, 82%) as a pale yellow solid.
Hexacyclic imine (+)-52 (2.2 mg, 15%) was also obtained as a yellow
solid byproduct. Structural assignment of (+)-52 utilized additional
information from gCOSY, HSQC, and HMBC. 1H NMR (500.4 MHz,
CD3OD, 21 °C): δ 7.63 (s, 1H, C25′H), 7.58 (d, J = 8.1 Hz, 1H, C7′H),
7.46 (d, J = 8.3 Hz, 1H, C4′H), 7.29 (dt, J = 7.6, 0.8 Hz, 1H, C6′H),
7.24 (d, J = 8.2 Hz, 1H, C18′H), 7.15 (d, J = 2.3 Hz, 1H, C15′H), 7.11
(t, J = 7.5 Hz, 1H, C5′H), 6.86 (dd, J = 8.2, 2.3 Hz, 1H, C17′H), 3.95
(app-dd, J = 13.3, 4.0 Hz, 1H, C11′H), 3.85 (s, 3H, OMe), 3.64−3.53
(m, 2H, C22′H2), 3.21 (app-t, J = 5.3 Hz, 2H, C10′H2), 3.19−3.12 (m,
1H, C11′H2), 1.82 (dt, J = 12.4, 5.1 Hz, 1H, C21′H), 1.36 (dd, J = 12.4,
3.4 Hz, 1H, C21′H), 2.96 (ddd, J = 16.9, 12.1, 4.3 Hz, 1H, C10′H),
μmol, 1.2 equiv) was added to the reaction mixture. After 9 min,
additional chloroform (80 μL) was added to the reaction mixture.
After 4 min, DIC (10.7 μL, 68.4 μmol, 1.20 equiv) was added to the
reaction mixture, and the resulting dark orange solution was placed in
a cryogenic cooler adjusted to 0 °C. After 22 h, the reaction mixture
was diluted with chloroform (0.5 mL) and directly purified by flash
column chromatography (silica gel: diameter 2.0 cm, ht. 11 cm; eluent:
13% ethyl acetate in hexanes) to afford hydroxyindolenine (+)-69 (23
mg, 97%) as a colorless oil. Hydroxyindolenine (+)-69 was found to
have 89.5:10.5 er by chiral HPLC analysis [Chiralpak IC 0.5 mL/min;
80% hexanes, 20% 2-propanol; tR(major) = 12.4 min, tR(minor) = 10.1
1
min]. H NMR (500.4 MHz, CDCl3, 21 °C): δ 8.15 (dd, J = 7.7, 1.4
Hz, 1H), 7.93 (dd, J = 8.1, 1.1 Hz, 1H), 7.66 (dt, J = 7.6, 1.3 Hz, 1H),
7.58 (appt-dt, J = 7.7, 1.5 Hz, 1H), 7.51−7.49 (m, 1H), 7.46−7.45 (m,
1H), 7.35 (dt, J = 7.6, 1.4 Hz, 1H), 7.27 (dt, J = 7.4, 1.1 Hz, 1H), 5.11
(s, 1H), 3.98 (ddd, J = 10.6, 7.9, 4.1 Hz, 1H), 3.77 (ddd, J = 10.7, 6.1,
4.6 Hz, 1H), 2.21 (ddd, J = 12.6, 8.0, 4.6 Hz, 1H), 2.03 (ddd, J =
10.26, 6.1, 4.2 Hz, 1H), 0.88 (s, 9H), 0.04 (s, 3H), 0.04 (s, 3H). 13C
NMR (125.8 MHz, CDCl3, 21 °C): δ 177.6, 152.9, 149.4, 140.5, 132.5,
130.9, 130.4, 129.8, 129.6, 127.1, 124.6, 123.0, 122.1, 89.1, 61.1, 37.4,
26.0, 18.2, −5.5, −5.6. FTIR (neat) cm−1: 3406 (s), 2955 (s), 2857
(m), 1649 (w), 1538 (s), 1471 (m), 1361 (m), 1258 (m), 1984 (m),
838 (s). HRMS (DART, TOF) (m/z): calcd for C22H29N2O4Si, [M +
H]+: 413.1891, found: 413.1890. [α]24D +136.1 (c 0.49, CHCl3). TLC
(25% ethyl acetate in hexanes), Rf: 0.43 (CAM, UV).
(R)-2-(2-((tert-Butyldimethylsilyl)oxy)ethyl)-2-(2-nitro-
phenyl)indolin-3-one (70). To a solution of hydroxyindolenine
(+)-69 (26.2 mg, 63.5 μmol, 1 equiv) in freshly distilled tert-butanol
(1.6 mL) under argon was added a solution of potassium tert-butoxide
(3.8 mg, 32 μmol, 0.50 equiv) in tert-butanol (1.6 mL) via syringe at
23 °C. After 16.5 h, saturated aqueous ammonium chloride solution (3
mL) was added and the resulting mixture was diluted with water (4.5
mL) and ethyl acetate (6 mL), and the layers were separated. The
aqueous layer was extracted with ethyl acetate (2 × 6 mL), and the
combined organic layers were dried over anhydrous sodium sulfate,
filtered, and concentrated under reduced pressure. The sample of the
crude residue was purified by flash column chromatography (silica gel:
diameter 2.0 cm, ht. 10 cm; eluent: 13% ethylacetate in hexanes) to
afford indoxyl (−)-70 (18 mg, 67%) as a yellow oil. Indoxyl (−)-70
was found to have 89.5:10.5 er by chiral HPLC analysis [Chiralpak IC
0.5 mL/min; 80% hexanes, 20% isopropanol; tR(major) = 14.9 min,
tR(minor) = 24.8 min]. 1H NMR (500.4 MHz, CDCl3, 21 °C): δ 7.79
(appt-d, J = 7.4 Hz, 1H), 7.61 (td, J = 7.7, 0.6 Hz, 1H), 7.53−7.50 (m,
1H), 7.48 (ddd, J = 8.3, 7.1, 1.3 Hz, 1H), 7.36−7.35 (m, 2H), 6.90
(appt-t, J = 7.8 Hz, 1H), 6.86 (td, J = 8.2, 0.7 Hz, 1H), 6.13 (s, 1H),
3.79−3.72 (m, 2H), 2.53 (ddd, J = 14.9, 6.8, 2.9 Hz, 1H), 2.08 (ddd, J
= 14.8, 9.7, 4.9 Hz, 1H), 0.84 (s, 9H), −0.09 (s, 3H), −0.10 (s, 3H).
13C NMR (125.8 MHz, CDCl3, 21 °C): δ 201.5, 160.8, 150.9, 137.7,
2.19−2.13 (m, 1H, C21′H), 2.06 (dd, J = 12.0, 5.8 Hz, 1H, C21′H). 13
C
NMR (125.8 MHz, CD3OD, 21 °C): δ 177.7 (C24′), 163.1 (C16′),
155.8 (C14′), 151.2 (C25′), 133.3 (C8′), 136.0 (C19′), 129.4 (C9′), 128.3
(C2′), 126.2 (C16′), 124.5 (C18′), 121.7 (C3′), 121.1 (C5′), 120.7 (C7′),
113.1 (C4′), 112.6 (C17′), 108.3 (C15′), 75.6 (C20′), 56.3 (C27′), 51.2
(C11′), 41.1 (C22′), 34.0 (C21′), 30.9 (C10′). FTIR (neat) cm−1: 3057
(br-s), 2929 (br-s), 1632 (s), 1581 (s), 1483 (s), 1334 (s), 1147 (s),
1027 (m), 735 (s). HRMS (ESI, TOF) (m/z): calcd for C22H21N4O,
[M + H]+: 357.1710, found: 357.1717. [α]D:24 +151 (c 0.43, MeOH).
TLC (9% methanol, 1% ammonium hydroxide in chloroform), Rf:
0.15 (CAM, UV).
(R)-2-(2-(3-(3-(2-(1,3-Dioxoisoindolin-2-yl)ethyl)-1H-indol-2-
yl)-6-methoxy-2-oxoindolin-3-yl)ethyl)isoindoline-1,3-dione
(62). Lanthanum(III) trifluoromethanesulfonate (22.7 mg, 38.7 μmol,
0.500 equiv) was added to a solution of hydroxyindolenines (+)-36
and (+)-38 (36:38, 2.2:1, 48.5 mg, 77.6 μmol, 1 equiv) in toluene (7.7
mL) at 23 °C, and the resulting reaction mixture was warmed to 80
°C. After 19.5 h, the reaction mixture was allowed to cool to 23 °C,
saturated aqueous sodium bicarbonate solution (15 mL) and ethyl
acetate (15 mL) were added and the layers were separated. The
aqueous layer was extracted with dichloromethane (15 mL). The
combined organic layers were dried over anhydrous sodium sulfate,
were filtered, and were concentrated under reduced pressure. The
residue was purified by flash column chromatography (silica gel:
diameter 2 cm, ht 10 cm; eluent: 3.3% acetone in dichloromethane to
10% acetone in dichloromethane) to afford indoxyls (−)-60 and
(−)-61 (60:61, 1:1.2, 27 mg, 56%) and oxindole (+)-62 (17 mg, 34%)
as a mixture of yellow solids. 1H NMR (500.4 MHz, CDCl3, 21 °C): δ
9.68 (s, 1H), 8.93 (s, 1H), 7.78 (dd, J = 5.3, 3.1 Hz, 2H), 7.70−7.65
(m, 5H), 7.61 (dd, J = 5.5, 3.1 Hz, 2H), 7.45 (d, J = 8.1 Hz, 1H),
7.16−7.05 (m, 3H), 6.60 (d, J = 2.3 Hz, 1H), 6.44 (dd, J = 8.2, 2.3 Hz,
1H), 4.06−4.00 (m, 1H), 3.97−3.91 (m, 1H), 3.69 (s, 3H), 3.64−3.51
(m, 2H), 2.83−2.76 (m, 2H), 2.70−2.64 (m, 1H), 2.57−2.51 (m, 1H),
2.71−2.66 (m, 1H), 2.37−2.32 (m, 1H). 13C NMR (125.8 MHz,
CDCl3, 21 °C): δ 179.6, 168.6, 168.3, 160.6, 142.1, 135.4, 134.2, 133.9,
132.3, 132.1, 131.6, 129.2, 125.3, 123.5, 123.5, 123.1, 122.3, 119.8,
118.7, 111.4, 109.8, 108.2, 98.3, 55.6, 51.6, 37.7, 34.7, 33.9, 23.9. FTIR
(neat) cm−1: 3335 (br-s), 3020 (m) 2361 (w), 1771 (m), 1709 (s),
1633 (m), 1397 (s), 1032 (w), 718 (s). HRMS (DART, TOF) (m/z):
calcd for C37H29N4O6, [M + Na]+: calculated: 647.1901, found:
131.0, 130.9, 129.7, 128.5, 125.3, 123.9, 120.6, 119.8, 113.7, 70.6, 60.1,
40.2, 26.0, 18.2, −5.7. FTIR (neat) cm−1: 3350 (m), 2954 (m), 2929
(m), 1705 (s), 1617 (s), 1533 (s), 1484 (m), 1369 (m), 1324 (m),
1258 (m), 1096 (s), 836 (s), 778 (s). HRMS (DART, TOF) (m/z):
calcd for C22H29N2O4Si, [M + H]+: 413.1891, found: 413.1903. [α]D
24: −79.5 (c 0.15, CHCl3). TLC (17% ethyl acetate in hexanes), Rf:
0.32 (CAM, UV).
Cell Culture Information. U-937 and HeLa cells were grown in
RPMI-1640 supplemented with fetal bovine serum (10%, FBS) and
antibiotics (100 μg/mL penicillin and 100 U/mL streptomycin). Cells
were incubated at 37 °C in a 5% CO2, 95% humidity atmosphere.
IC50 Value Determination for HeLa Cells Using Sulforhod-
amine B (SRB). HeLa cells were added into 96-well plates (1,500
cells/well) in 100 μL media and were allowed to adhere for 2−3 h.
Compounds were solubilized in DMSO as 100× stocks, added directly
to the cells (100 μL final volume), and tested over a range of
concentrations (0.25−100 μM) in triplicate (1% DMSO final) on a
third-log scale. DMSO and cell-free wells served as the live and dead
control, respectively. After 72 h of continuous exposure, the plates
were evaluated using the SRB colorimetric assay as described
previously.55 Briefly, media was removed from the plate, and cells
were fixed by the addition of 100 μL cold 10% trichloroacetic acid in
647.1914. [α]24 +93.6 (c 0.24, CHCl3). TLC (14% acetone in
D
dichloromethane), Rf: 0.34 (CAM, UV).
(R)-3-(2-((tert-Butyldimethylsilyl)oxy)ethyl)-2-(2-nitrophen-
yl)-3H-indol-3-ol (69). To a glass vial charged with tryptophol
derivative 68 (22.6 mg, 57.0 μmol, 1 equiv) and catalyst 40 (3.7 mg,
5.7 μmol, 0.10 equiv) was added DMAP (0.35 mg, 2.9 μmol, 0.050
equiv) in chloroform (200 μL), and the reaction mixture was cooled to
0 °C. After 3 min, hydrogen peroxide (30% w/w in water, 7.0 μL, 68
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The Journal of Organic Chemistry
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water. After being incubated at 4 °C for 1 h, the plates were washed in
water and allowed to dry. Sulforhordamine B was added as a 0.057%
solution in 1% acetic acid (100 μL), and the plates were incubated at
room temperature for 30 min, washed in 1% acetic acid, and allowed
to dry. The dye was solubilized by adding 10 mM Tris base solution
(pH 10.5, 200 μL) and incubating at room temperature for 30 min.
Plates were read at λ = 510 nm.
IC50 Value Determination for U-937 Cells Using MTS. In a 96-
well plate, compounds were preadded as DMSO stocks (1% final) in
triplicate to achieve final concentrations of 0.25 μM to 100 μM on a
third-log scale. DMSO and cell-free wells served as the live and dead
control, respectively. U-937 (5,000 cells/well) cells were distributed in
100 μL media to the compound-containing plate. After 72 h, cell
viability was assessed by adding 20 μL of a PMS/MTS solution56 to
each well, allowing the dye to develop at 37 °C until the live control
had processed MTS, and reading the absorbance at λ = 490 nm.
(12) Buyck, T.; Wang, Q.; Zhu, J. Org. Lett. 2012, 14, 1338−1341.
(13) Qiu, J.; Zhang, J.-X.; Liu, S.; Hao, X.-J. Tetrahedron Lett. 2013,
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(15) (a) Zhao, B.; Hao, X.-Y.; Zhang, J.-X.; Liu, S.; Hao, X.-J. Org.
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( )-trigonoliimine C, see: Reddy, B. N.; Ramana, C. V Chem.
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(16) For elegant examples of the application of oxidation and
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D.; Sanz-Cervera, J. F.; Williams, R. M. J. Am. Chem. Soc. 1996, 118,
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E. J. J. Am. Chem. Soc. 2002, 124, 7904−7905. (i) Liu, Y.; McWhorter,
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2003, 5, 333−335. (l) Baran, P. S.; Richter, J. M. J. Am. Chem. Soc.
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ASSOCIATED CONTENT
* Supporting Information
■
S
General procedures, supplementary discussion, and copies of
1H and 13C NMR spectra and HPLC data. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: movassag@mit.edu.
■
Notes
The authors declare no competing financial interest.
(17) Schmidt, M. A.; Movassaghi, M. Synlett 2008, 313−324.
(18) See the Supporting Information for details.
(19) Movassaghi, M.; Schmidt, M. A.; Ashenhurst, J. A. Org. Lett.
ACKNOWLEDGMENTS
■
We acknowledge financial support by NIH-NIGMS
(GM074825) and the University of Illinois. S.H. acknowledges
EMD Serono and Kenneth M. Gordon summer graduate
fellowships. K.C.M. is a National Science Foundation
predoctoral fellow and a Robert C. and Carolyn J. Springborn
graduate fellow. We thank Changkyu Han for the sketch of the
Trigonostemon lii Y. T. Chang used in the cover image.
2008, 10, 4009−4012.
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