Six Trikentrin-like Cyclopentanoindoles from Trikentrion flabelliforme. Absolute Structural Assignment by NMR and ECD

Article abstract of DOI:10.1021/acs.joc.7b02813

Six new cyclopenta[g]indoles were isolated from a West Australian sponge, Trikentrion flabelliforme Hentschel, 1912, and their structures elucidated by integrated spectroscopic analysis. The compounds are analogues of previously described trikentrins, herbindoles, and trikentramides from related Axinellid sponges. The assignment of absolute configuration of the new compounds was carried out largely by comparative analysis of specific rotation, calculated and measured ECD, and exploiting van't Hoff's principle of optical superposition. Five of the new compounds were chemically interconverted to establish their stereochemical relationships, leading to a simple chiroptical mnemonic for assignment of the this family of chiral indoles. The first biosynthetic hypothesis is advanced to explain the origin of the trikentrin-herbinole family and proposes a pyrrole-carboxylic thioester-initiated polyketide synthase mechanism.

Full text of DOI:10.1021/acs.joc.7b02813

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Article
Six Trikentrin-like Cyclopentanoindoles from Trikentrion
flabbeliforme. Absolute Structural Assignment by NMR and ECD.
Mariam N Salib, and Tadeusz F Molinski
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 10 Jan 2018
Downloaded from http://pubs.acs.org on January 10, 2018
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Six Trikentrin-like Cyclopentanoindoles from Trikentrion
flabbeliforme. Absolute Structural Assignment by NMR and
ECD
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†
,†,§
Mariam N. Salib and Tadeusz F. Molinski*
†Department of Chemistry and Biochemistry, and
§Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California,
San Diego,
9
500 Gilman Dr. MC-0358, La Jolla, California 92093-0358.
*
To whom correspondence should be addressed: Tel: +1 (858) 534-7115. Fax: +1 (858) 822-0386.
E-mail: tmolinski@ucsd.edu
ꢀ
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ABSTRACT: Six new cyclopenta[g]indoles were isolated from a West Australian sponge,
Trikentrion flabbeliforme Hentschel, 1912 and their structures elucidated by integrated
spectroscopic analysis. The compounds are analogs of previously described trikentrins,
herbindoles and trikentramides from related Axinellid sponges. The assignment of absolute
configuration of the new compounds was carried out largely by comparative analysis of specific
rotation, calculated and measured ECD and exploiting van’t Hoff’s principle of optical
superposition. Five of the new compounds were chemically interconverted to establish their
stereochemical relationships, leading to a simple chiroptical mnemonic for assignment of the this
family of chiral indoles. The first biosynthetic hypothesis is advanced to explain the origin of the
trikentrin-herbinole family, and proposes a pyrrole-carboxylic thioester initiated polyketide
synthase mechanism.
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Introduction
The majority of marine indole alkaloids are derived from secondary metabolism of
tryptophan. The indigo derivative, ‘Tyrian purple’ (6,6’-dibromoindigotin), from the
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1
hypobranchial gland of various marine mollusks, has been known since ancient times:
2
contemporary studies revealed its structure through X-ray crystallography. Still more rare are
simple indoles that lack carbon substitution at C-3; their structures imply biosyntheses that
diverge substantially from tryptophan metabolism. In a study by Brennan and Erickson, one of
the first investigations of red algae, 10 achiral polyhalogenated indoles were reported from
Rhodophyllis membranacea Harvey, and more recent studies of R. membranacea have delivered
3
another 18 new analogs.
Among the more curious natural indoles are unique, optically active cyclopenta[g]indoles
from two Axinellid sponges. Capon and MacLeod described antibacterial (+)-cis- and (+)-trans-
trikentrins A (1, 2), (–)-trans-trikentrin B (4) and an inseparable mixture of cis- and iso-trans-
trikentrin B (3 and 5, respectively) from Trikentrion flabbeliforme Hentschel, 1912, collected in
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Darwin, Australia. Scheuer and coworkers characterized the trikentrin homologs, (–)-
herbindoles A-C (6–8), from a specimen of Axinella sp. collected in Exmouth Gulf, Western
5
Australia. The latter compounds, which exhibit modest cytotoxicity and fish antifeedant activity,
are antipodal in configuration to the cis-trikentrins as established through independent total
6
7
syntheses of ent-(–)-1, (+)-2, and ent-(+)-6–8 by Natsume and coworkers, and of (+)-4 by
8
Kanematsu and coworkers. (+)-Trikentramides A-D (9–12), isolated and characterized by Quinn
and coworkers from a specimen of T. flabbeliforme collected at Port Hedland (500 km from
9
Exmouth), are isatin and oxindole analogs of (+)-cis- and (+)-trans-trikentrins A.
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N
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N
H
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R
N
H
N
H
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: R = H
: R = Me
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R
O
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H
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: R = H
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1
1: R = Me
O
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R
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O
O
5a
8b
N
H
N
H
N
H
8
a
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1
3
14
15: R = H
6: R = Me
1
OH
OH
O
O
N
H
N
H
1
7
18
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10,11
12
Aside from the above-referenced syntheses, the total syntheses of (+)-1,
(+)-2, (±)-1
1
1,13,14,15,16,17,18
11
13,19
13,19,20
and (±)-2,
(+)-3, (±)-3,
and (±)-6–8 have been achieved.
A formal
2
1
synthesis of (±)-3 has also been reported, and all total syntheses of trikentrin-herbindole family,
2
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up to 2009, have been reviewed.
In all likelihood, the biosynthesis of polyhalogenated indoles in red alga is linked to the
enduring natural history of indole, indoxyl, indigo dye, and other indigoid compounds from
plants of the genus Indigofera and, as shown through genetic and ecological studies, bacterial
23
biosynthesis.
On the other hand, the origin of the sponge-derived 1,3-
dimethylcyclopenta[g]indoles must differ considerably from the latter. To date, no hypotheses
for the origin of 1–12 have been advanced.
Here, we report the structures of new trikentrin-like natural products, (+)-trans-
herbindole A (13) and trikentramides E-I (14–18), from T. flabbeliforme collected in Exmouth
Gulf. The absolute configurations of 13–18 were assigned from chiroptical comparisons with
24
trikentrins and herbindoles, and exploitation of van’t Hoff’s principle of optical superposition,
and chemical correlation to derive a new mnemonic for absolute stereostructure of this family of
compounds. Surprisingly, the structures of 13–18 uniformly exhibit a trans configuration for the
fused 1,3-dimethylcyclopentane ring, in contrast with the (–)-cis-herbindoles (6–8) obtained
4
from a specimen of T. flabbeliforme collected in the same vicinity, and are enantiomorphic with
the latter. A unifying hypothesis is proposed that rationalizes the biogenesis of trikentrins,
herbindoles, trikentramides and two related sponge-derived indoles from West African
Trikentrion loeve.
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Results and Discussion.
A MeOH extract of lyophilized Trikentrion flabbeliforme, collected in Exmouth Gulf,
Western Australia in 1993, was redissolved in MeOH-H O (9:1), and the solution was partitioned
2
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progressively against hexanes, CH Cl , and n-BuOH. The CH Cl -soluble ‘fraction B’, enriched
2
2
2
2
in indoles (TLC spots stained bright fuschia with vanillin-H SO ), was separated by gel filtration
2
4
chromatography (Sephadex LH-20) and further purified by reversed-phase HPLC (C ) to obtain
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pure 13 –18.
The simplest of the new compounds, trans-herbindole A (13), C H N (HRESITOFMS
1
5
19
+
m/z 214.1592 [M+H] ), when compared with cis-herbindole A (6), confirmed that 13 and 6 are
constitutional isomers, but diastereomeric in the 1,3-dimethylcyclopentane ring. Compound 13
1
displayed a relatively featureless H NMR spectrum (CDCl ) consisting of signals for a trans-
3
1,3-dimethylcyclopentane ring: two methyl doublets (δ 1.21, 3H, d, J = 6.6 Hz and δ 1.49, 3H,
H
H
d, J = 7.2 Hz), two methines (δ 3.46, 1H, quintet, J = 7.2 Hz; δ 3.71, 1H, qdd, J = 7.2, 7.8, 9.6
H
H
Hz) and H -7, a diastereotopic methylene group (H-7α δ 2.10, 1H, ddd, J = 1.2, 7.2, 12.2 Hz
2
H
2
5
and H-7β, δ 1.97, 1H, ddd, J = 7.8, 9.6, 12.0 Hz). The relatively small separation in chemical
H
shifts of the H -7 proton signals (Δδ = 0.14 ppm) is characteristic of pseudo-enantiotopic
2
methylene protons in trans-1,3-dimethylcyclopenta[g]indole rings (e.g. enantiotopic CH in
2
trans-1,3-dimethylindane, Figure 1a), as opposed to the larger signal separation (Δδ >1.3 ppm)
5,9
observed in the corresponding diastereotopic methylene of the cis-isomers.
A broad
exchangeable singlet (δ 8.02, 1H, bs) was assigned to the indole NH. The remaining signals were
pairs of aryl methyls (δ 2.35, 3H, s and δ 2.50, 3H, s), and pyrrole ring proton signals (δ 6.55,
H
H
H
13
9
1
H, s and δ 7.14, 1H, s). Comparisons of the C NMR chemical shifts of 13 with those of 9-12
H
5
and cis-herbindole A (6) further supported a 4,5-dimethylindole structure.
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Trikentramide E, C H NO, (14) is an oxindole; the higher homolog of 12. The C
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5
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chemical shifts of 14 at C-2 (δ 177.7 Cq) and C-3 (δ 35.7, CH ) compare well with those of
C
C
2
2
6
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indolin-2-one, but not indolin-3-one. Again, the 1,3-dimethylindane ring is trans-substituted.
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(a)
(b)
H
Me
H
Me
H
Me
H
Ha
Hb
Ha
1
3
Me
Hb
trans-i
cis-ii
Figure 1. Stereotopicity of the CH group in trans- and cis-1,3-dimethylcyclopentanobenzene
2
(
1,3-dimethylindane). (a) enantiotopic (b) diastereotopic.
The formulas of trikentramides F (15, C H NO ) and G (16, C H NO ), with two
14 15
2
15 17
2
oxygens, suggested homologous isatins, differing by substitution by Me at C-5 in 16. The red-
shifted long-wavelength band in the UV-vis spectrum of each compound [λmax 326 (log 3.54)]
10
1
3
was consistent with a substituted isatin (indolin-2,3-dione). The C NMR spectra (CDCl )
3
showed pairs of downfield signals attributed to the aryl ketone and lactam carbonyl groups,
respectively [e.g. 16, δ 183.2 (C-3), 160.2 (C-2)], matched those reported for the parent
2
8
1
heterocycle, isatin (δ 184.5, 159.5, DMSO-d ). The H NMR spectra of the two compounds
6
appeared similar to those of 14 and supported the same trans-1,3-dimethylcyclopenta[g]indole
1
13
ring. Full H and C NMR assignments (Tables 1 and 2) were secured through analysis of
COSY, NOESY, HSQC and HMBC spectra (Figures 2 and 3).
The formula of isomeric dioxindoles (+)-trikentramides H (17) and (+)-I (18),
+
+
C H NO Na (HRESITOFMS m/z 268.1307 and m/z 268.1305, respectively [M+Na] )
1
5
19
2
1
3
confirmed the two are formal reduction products of 16. The C chemical shifts of 18 at δ 178.9
C
3
0
(
C-2) and 69.7 (C-3), which are consistent with those of 3-hydroxy-5-methyl-indolin-2-one,
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verified the dioxindole arrangement of keto and hydroxymethine groups at C-2 and C-3,
respectively.
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O
OH
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H
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COSY
HMBC
Figure 2. COSY and HMBC correlations of (–)-trikentramide G (16) and (+)-trikentramide I
3
(18) (500 MHz, CDCl ).
O
O
N
H
H
H
1
6
Figure 3. NOESY correlations of (–)-trikentramide G (16) (600 MHz, CDCl3).
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1
a
Table 1. H NMR Data (δ, mult) for 13–18.
#
13
14
15
16
17
18
1
1
1
1
1
1
1
1
1
1
2
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2
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5
5
5
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0
1
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1
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9
0
NH
2
8.03, bs
7.14, t, 2.4
7.97, bs
8.80, bs
8.96, bs
7.99, bs
4.98, s
8.21, bs
4.99, s
6.55, dd (2.4, 3.40, s
3
3
.0)
4
5
6.68, s
3.27, m
b
b
c
b
b
c
3
7.2)
.46, quin
3.31, quin
(7.2)
3.29, quin 3.27, quin
(7.2, 0.9) (7.2)
3.30, m
6
(
d
2.10, ddd,
2.01, ddd, 2.02, ddd, 2.04, ddd, 1.97, dd,
(1.2, 7.8,
12.3)
1.83, ddd, 1.91, dt,
(7.2, 9.9,
12.3)
3.43, qdd, 3.31, m
(6.6, 7.8,
9.9)
2.18, s
2.14, s
1.96, bdd ,
7
ꢀ
ꢁ
(1.2, 7.5,
2.1)
.97, ddd,
(7.2, 9.6,
2.1)
(3.0, 7.8,
12.6)
(1.5, 8.2,
12.6)
1.85, ddd, 1.79, dt,
(7.8, 12.6) (7.8, 12.6)
1
1
1.79, m
3.40, m
7
(8.4, 12.6) (7.2, 9.6, (9.0, 12.6)
1
12.6)
c
c
3.44, qdd, 3.37, m
(7.2, 8.2,
9.6)
2.49, s
2.14, s
1.14, d,
(7.2)
1.36, d,
(7.2)
8
3.71, qdd,
(7.2, 7.5, 9.6)
4
5
-Me
-Me
2.50, s
2.35, s
2.55, s
2.28, s
2.15, s
1.12, d,
(7.2)
1.27, d,
(6.0)
2.28, s
2.15, s
1.07, d,
(7.2)
1.25, d,
(6.6)
1
.13, d,
.21, d, (6.6) (6.6)
1.32, d,
.49, d, (7.2) (6.6)
1.26, d,
(7.2)
6
-Me
1
1.23, d,
(6.6)
8
-Me
1
a
b
c
d
6
00 MHz, CDCl
3
. quin = quintet. overlap. bdd = broad doublet of doublets.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13
a
Table 2. C NMR Data (δ, mult) for 13–18 (CDCl , 125 MHz).
3
14
15
16
17
18
#
2
3
13
122.9, CH
101.6, CH
126.3, C
128.1, C
123.1, C
125.7, C
38.4, CH
177.7, C
35.7, CH2
123.2, C
135.2, C
125.9, C
148.7, C
37.9, CH
160.3, C
160.2, C
183.2, C
115.7, C
139.9, C
127.0, C
160.6, C
38.8, CH
178.5, C
69.7, CH
124.1, C
134.6, C
127.1, C
150.9, C
37.9, CH
178.9, C
69.7, CH
124.1, C
134.4, C
127.3, C
150.8, C
38.0, CH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
b
3
a
115.2, C
140.9, C
121.0, C
161.4, C
38.5, CH
4
5
5
a
6
7
8
43.9, CH2
43.8, CH2
43.0, CH2
43.2, CH2
43.8, CH2
43.7, CH2
36.5, CH
35.4, CH
34.6, CH
35.2, CH
35.6, CH
35.5, CH
8
a
143.0, C
130.8, C
125.3, C
131.5, C
16.1, CH3
16.1, CH3
19.7, CH3
20.1, CH3
128.9, C
144.5, C
18.5, CH3
–
128.4, C
142.8, C
14.5, CH3
13.4, CH3
19.5, CH3
19.6, CH3
125.7, C
134.3, C
15.4, CH3
14.9, CH3
19.8, CH3
126.0, C
134.1, C
15.4, CH3
14.9, CH3
19.6, CH3
19.9, CH3
8
b
4-Me 15.7, CH3
5
6
-Me 15.2, CH3
-Me 20.4, CH3
19.3, CH3
19.8, CH3
8-Me 20.7, CH3
20.0, CH3
a
b
Multiplicities determined from edited HSQC, or comparisons with 9–12. MHz (Ref. 9). Signal not observed.
Absolute configurations of 13–18 were assigned from analysis of the CD and [α]
and comparisons with published chiroptical data of known compounds and DFT calculations of
ECD. The simplest assignment, by comparisons of [α] s (Table 3), is that of (+)-trans-
D
data,
D
herbindole A (13, [α]
D
+29.0) which follows from its structural similarity to (+)-trans-trikentrin
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D D
A (2, [α] +23.3) and the relative invariance of [α] in trikentrins and herbindoles A and B,
whether substituted at C-4 or C-5 by H, Me or Et (Table 3); therefore, both compounds bear the
same (6S,8S) configuration (see below for more discussion).This is not necessarily true of 3–5,
and 8; the conjugated E-1-butenyl chain, which constitutes a substituted styrenyl chromophore,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
D
adds complexity to the [α] of these indoles and abrogates simple chiroptical comparisons.
The specific rotations of oxidized indoline analogs, trikentramides A-I, do not follow a
simple pattern; the magnitude and sign vary with substitution and electronic properties in a
complex manner. For example, the two isatins 15 and 16 differ only in a single Me group at C-5,
but show [α]
D
s ~0 and –43.4, respectively. These are confoundingly different from 13 and 14
of highly dextrorotatory 18 (+232) appears to
(+29 and +19.0, respectively). Moreover, the [α]
D
be largely influenced by the stereocenter at C-3. Consequently, in order to make the
stereoassignments of 15 and 16, we first chose to assign 17 and 18 by ECD and chemically
correlate them to other family members.
Assignment of absolute configuration of trikentramides H (17) and I (18), which are
epimeric dioxindoles, rested upon interpretation of their corresponding ECD spectra (Figure 4).
The ECD spectra of oxidized indoles 14–16 exhibited relatively weak Cotton effects (CEs), not
7
unlike Natsume’s observations of indoles 1-3. In contrast, the ECDs of 17 [λ 243 (Δε –7.4), 221
(+8.6)] and 18 [λ 243 (Δε +5.0), 224 (–5.6)] are virtual mirror images dominated by relatively
strong biphasic CEs that reveal a predominant influence of the C-3 stereocenter. Asymmetric
reductions of isatins to 3-hydroxoxindoles have been reported that provide comparison
2
9,30,31
compounds for chiroptical assignments of 17 and 18.
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1
1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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5
5
5
5
5
5
5
5
5
6
O
OH
H , 5 bar
2
O
O
N
H
Pt-Al O
2
3
N
H
(–)-cinchonidine
dioxane, rt
2
0
19
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 1. Partial asymmetric hydrogenation of 5-methylisatin. See Ref. 30 and Supporting
Information for ECD.
(
a)
(b)
(
c)
(d)
Figure 4. ECD spectra (CH CN, 23 ˚C) of (a) trikentramides H [(+)-17] and I [(+)-18], dash). (b)
trikentramides F [15] and G [(–)-16, dash]. (c) trikentramides F [(–)-16] and E [(+)-14, dash]. (d)
trans-herbindole A [(+)-13].
3
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9
For example, isatin undergoes enzyme-catalyzed reduction in the presence of a carbonyl
3
1
reductase derived from Candida parapsilosis to give (+)-(R)-dioxindole in 21%ee. (R)-5-
Methyldioxindole (19, 38.5%ee), prepared by Sonderegger and coworkers using partial
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2 2 3
asymmetric hydrogenation of 20 (H , Pt supported on Al O -(–)-cinchonidine, Scheme 1),
exhibited an ECD spectrum dominated by a negative Cotton effect (CE) (λ 237 nm, –41,
arbitrary units of ellipticity) and a second CE of opposite sign at longer wavelength (λ 264,
30
+
12.5). The authors calculated the ECD spectrum of 19 using time dependent DFT (b3pw91
3
2
and 6-31G(d, p) basis set ) and assigned their reduction product (R)-19. We repeated the
reduction of 20 under non-asymmetric conditions (EtOH, NaBH ) and resolved the product, (±)-
9, by chiral phase HPLC (Phenomenex Lux 5U, Amylose 2) into (S)-19 and (R)-19 which, as
4
1
expected, exhibited mirror image ECD spectra (see Supporting Information). The near identity of
the ECD spectra of (R)-19 and (+)-18, and the expected weaker dichroic contributions from the
1
,3-dimethylcyclopentane in other trikentramides (Figure 4b,c), leads to the conclusion that the
configuration of the natural products are (3S,6S,8S)-17 and (3R,6S,8S)-18. Reduction of 16
(Scheme 2, NaBH
4
, EtOH, rt) gave the C-3 epimeric dioxindoles 17 and 18, identical with the
1
natural products by HPLC retention times, H NMR, [α]
D
and ECD. In addition, a minor
product, from over-reduction of intermediate 17/18 at the benzylic carbon, was recovered and
1
shown to match (+)-trikentramide E (14) by HPLC retention time, H NMR and ECD. Finally,
oxidation of the indole (+)-13 under the selective conditions recently reported by Wang and
33
2
coworkers (I , t-BuOOH, DMSO, 80 ˚C, Scheme 2), gave a product identical with natural (–)-
1
1
6 by MS, H NMR, ECD and HPLC retention time. Thus, (+)-trans-herbindole A (13) is
chemically correlated to (+)-14, (–)-16, (+)-17 and (+)-18 demonstrating that the
dimethylcyclopentane ring is stereochemically uniform among these five natural products.
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
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3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Although we have no independent correlation of (+)-trikentramide E (15), it seems highly
3
4
probable the configuration is the same as to its congeneric family members.
The observed ECD spectra of 17 and 18 are the molar sum contributions of independent
asymmetric perturbations to the benzenoid chromophore by the 1,3-dimethylcyclopentane group
and the benzylic secondary OH group within their respective ‘first spheres of asymmetry’. The
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4
latter is a formal corollary of van’t Hoff’s principle of optical superposition, which has been
applied both to configurational assignments through deconvolution of both molar rotation [M]
35
D
and ECD (Δε), in combination with DFT calculations (e.g. CADPAC calculated [α] of (–)-
3
5c
1
,3,5,7-tetramethyl-1,3-dihydroindol-2-one ).
O
OH
OH
O NaBH , rt
O
4
O
N
H
N
H
N
H
EtOH, 1h
(–)-16
(+)-17
(+)-18
I2, t-BuOOH,
DMSO, 80 ˚
O
N
H
N
H
(+)-14
(+)-13
Scheme 2. Chemical interconversions of (+)-13 with (+)-14, (–)-16, (+)-17 and (+)-18.
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Table 3. [α] for Natural and Synthetic Trikentrins, Herbindoles and Trikentramides.
D
a
b
Config.
Cmpd.
(+)-1
Source
N
S
[α]
+48
+49
Ref.
D
c
4
11
(+)-1
ent-
(–)-1
S
N
S
–68.6 6a,b
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
d
(+)-2
(+)-2
(+)-2
+23.3
+24
4
6c
10b
S
+24
ent-
ent-
(–)-2
(+)-3
S
S
S
S
N
S
S
N
S
S
–26.8 6a,b
+102
6c
8
11
4
(
(
+)-3
+)-3
+100.2
+101
e
(
–)-4
–13
(+)-4
5
(–)-6
(+)-6
(+)-7
(+)-8
+24
~0
–62
+56.9
6c
6c
7
ent-
ent-
ent-
7
+51.2 6a
S
+19.9
+40.7
+42.2
+50.0
+29.0
+19.0
~0
7
9
9
9
f
(+)-9
N
N
N
N
N
N
N
N
(
+)-11
(+)-12
(+)-13
(
+)-14
f
f
1
5
(
(
(
–)-16
+)-17
+)-18
–43.4
+22.6
+231.5
f
f
f
N
a
b
N = natural; S = synthetic. All measurements were made on solutions in
CHCl and, aside from the noted exceptions, within the concentration range c.
3
3
c
d
e
f
~0.027 – 0.50 g/100 cm . c = 2.47. c = 1.0. c = 1.97. This work.
The largest contribution to both the [α] and CD in trans- and cis-fused trikentrin-type
D
indoles appears to be the C-8 stereocenter. For example, both (+)-ent-cis-6 and (+)-trans-13 have
the same C-8 configuration and are dextrorotatory, although the magnitude of [α] of the former
D
is almost twice that of the latter (Table 3). The presence of a C-5 Me group has little effect on
[
α] ; trans-(+)-2 and (+)-13 have almost the same specific rotation ([α] = +23.3 and +29.0,
D
D
respectively). A brief conformational analysis of cis and trans herbindoles was carried out
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
3
4
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
MMFF94 and DFT) to derive their lowest energy conformations and frontier molecular orbitals.
The conformation of the cyclopenta[g]indole ring in 13 (Figure 5) is distorted from an idealized
C conformation by torsional strain and steric repulsion between the N-H bond and the methyl
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
group, and between the Me groups at C-5 and C-6. For example, the C-5–C-5a–C-6–C-6(Me)
dihedral angle is θ = 52.1˚, but the C-8b–C-8a–C-8–C-8(Me) dihedral angle is considerably
larger, ψ = 72.3˚, but the ‘unstrained’ trans-1,3-dimethylindane i – Figure 1 – which freely
inverts between the two degenerate C conformers – displays θ = 40.1˚; ψ = 76.1˚. In contrast,
2
the cyclopentane ring of (+)-cis-herbindole A (ent-6) has an almost symmetric envelope
conformation (θ = –53.2˚, ψ = 48.3˚).
The major difference in frontier molecular orbitals of the delocalized π-system in 1,3-
cyclopenta[g]indoles is the distortion of the LUMO by non-bonded interactions, particularly by
the C-8 methyl group, which influences the rotational strength, and therefore ECD and [α] , by
D
altering molecular orbital symmetry properties. The influence of the C-8 stereocenter upon
chiroptical properties is extrapolated into a simple stereochemical mnemonic (Figure 6): for
trikentrin-herbindole like indoles, the absolute configuration at C-8 determines the sign of [α]D,
whether the 1,3-dimethylcyclopentane ring is cis- or trans-fused. The exceptions are 1-pentenyl
substituted compounds (e.g. 3) where contributions from the additional rotational strength of the
styrenyl chromophore perturbs this simple rule. As the presence of C-4 or C-5 alkyl substituents
(e.g. Me, Et) appear not to significantly change the relationship between sign of specific rotation
and absolute configuration, we predict that hydrogenation of 1-pentenyl to an n-pentyl group,
prior to measuring the [α] , will restore the relationship; the products should conform to the
D
mnemonic and allow stereoassignment.
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(
a)
5
8
b
5
a
6
8
-Me
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
-Me
6
-Me
(b)
5
5
c
5
c
5
a
5b
8
6
-Me
6
-Me
8-Me
6
8-Me
Figure 5. Energy minimized geometries (MMFF) and DFT calculated (ωB97X-D 6-31G*)
LUMO of (a) (+)-trans-herbindole A (13) and (b) (+)-cis-herbindole A (ent-6).
Conformational searching returned only one conformation for 6 or 13.
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
3
4
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4
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4
4
5
5
5
5
5
5
5
5
5
5
6
3
3
R1
R1
R2
NH
R2
NH
H
Me
Me
H
Me
Hβ
Hβ
Hα
6
8
6
8
Me
H
H
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Hα
(a)
trans-
[α]D
cis-
[α]D
3
3
R1
R1
R2
NH
R2
NH
H
H
Me
H
H
Hβ
Hα
Hβ
Hα
6
8
6
8
Me
Me
Me
[α]D
(b)
trans-
[
α]D
cis-
Figure 6. Chiroptical mnemonic for assignment of absolute stereostructures of
trikentrins and herbindoles (a) trans- and cis-8S (b) trans- and cis-8R (R , R = H, alkyl,
1
2
but ≠ 1-alkenyl)
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The biosynthesis of most natural product indoles follows familiar routes. Free indole
is oxidatively liberated from Trp by the action of a tryptophanase (L-Trp indole lyase).
Certain bacteria are known to harbor the enzyme and, in combination with oxidases, could
give rise to polyhalogenated indoles in Rhodophycae. In considering possible biosynthetic
pathways that generate 1–18, degradation of Trp seems unlikely; not only does C-3 of the
indole ring lack a carbon substituent, but the introduction of the fused carbocyclic ring at
C-5a,b and additional carbon substituents at C-4 and C-5 necessitates unwieldy complexity
in this putative biogenesis. Consequently, alternative pathways must be considered.
Alignment of the structures of two C-2 substitued indenyl-pyrrole metabolites,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
36
37
trikentramine (21) and trikendiol (22) (Figure 7, both isolated from West African
Trikentrion loeve) with cis-trikentrin A (1) suggests a hypothesis: the trikentrin-herbindole
family are of polyketide origin. Proline is oxidatively modified to the hypothetical pyrrole-
2
-carboxylic as a starter unit for ketide extensions by malonate (acetate) or methylmalonate
3
8
(
propionate). In this scheme, the origin of the benzenoid ring differs slightly between two
pathways, a and b. Because the C-C bond formation aligns carbons formally derived from
the C=O group of the ketide extender units, it is unlikely that cyclization proceeds by
aldol/Claisen condensations familiar from the biosynthesis of aromatic Type II polyketides.
The indane ring of trikentramine (pathway a) is mostly likely assembled after partial
reductions and polyene electrocyclization reactions – possibly involving oxidative single
39
electron transfer (SET) – followed by aromatization to the benzene ring. For trikentrin-
like secondary metabolites, (pathway b), similar reactions proceed, but for the fused indole
ring we invoke a Friedel-Craft type intramolecular alkylation at C-3 (pyrrole numbering) of
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5
5
5
5
5
5
5
6
the electron-rich pyrrole ring for construction of the C-3b–C-4 bond. Indeed, Natsume
exploited a similar reaction for the indole ring-closing steps in the total syntheses of 2, 4,
and 5.6
0
1
2
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7
8
9
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HO
O
X
HN
5
4
NH
O
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
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3
4
4
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5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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8
9
0
1
2
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8
9
0
1
2
3
4
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8
9
0
NH
OH
Trikendiol (22)
O
Trikentramides
e.g. 12 (X = H)
<O>
<O>
X
5
4
3
b
NH
NH
Trikentramine (21)
cis-Trikentrin A (1),
X = H
Friedel-Crafts
rxn?
X
•
•
•
•
•
•
•
•
•
•
•
N
N
H
H
O
•
SR
N
pathway a
H
pathway b
Pro
X X = H, acetate
•
= CO
Me, propionate
Et, butyrate
•
pentenoate ( lost as CO2 )
•
Figure 7. Hypothesis for the biogenesis of trikentrin-like natural products, including cis-
trikentrin A (1), trikentramine (21) and trikendiol (22).
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1
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The hypothesis succinctly accounts for the location of substituents at C-4 and C-5,
namely H, Me, Et or 1-butenyl, through chain extensions or termination of the polyketide
chain by homologated malonate units. Subsequently, a manifold of oxidative reactions of
the product indoles would generate 20 or, conversely, the oxindole, isatin, and dioxindole
scaffolds found in trikentramides A–D (9-12) and E–I (14–18).
0
1
2
3
4
5
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9
0
1
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9
0
1
2
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Conclusion.
Six new cyclopenta[g]indole natural products – trans-herbindole A (13) and
trikentramides E-I (14–18) – were isolated from the sponge Trikentrion flabbeliforme and
their absolute stereostructures solved by integrated analysis of MS, NMR and ECD studies,
in combination with calculated DFT, and chemical correlation. A simple mnemonic
emerged from analysis of the chiroptical data that allows easy identification of absolute
configuration of indoles and dioxindoles within the series.
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured on a JASCO
P-2000 at the D-double emission line of Na. UV-vis spectra were measured on a JASCO
V-630, spectrometer. ECD spectra were measured on a JASCO J-810 spectropolarimeter in
quartz cells (1 or 5 mm pathlength) at 23 ˚C. FTIR spectra were collected on thin film
samples using a JASCO FTIR-4100 fitted with an ATR accessory (ZnSe plate). Inverse-
detected 2D NMR spectra were measured on a JEOL ECA (500 MHz) spectrometer,
1
13
equipped with a 5 mm H{ C} room temperature probe, or a Bruker Avance II (600 MHz)
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1
13 15
13
NMR spectrometer with a 1.7 mm H{ C/ N} microcryoprobe. C NMR spectra were
measured on a Varian NMR spectrometer (125 MHz) equipped with a 5 mm Xsens
1
3
1
C{ H} cryoprobe. NMR spectra were referenced to residual solvent signals, (CDCl , δ
H
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
.26, δ
C
77.00 ppm). High-resolution ESITOF analyses were carried out on an Agilent
1
200 HPLC coupled to an Agilent 6350 TOFMS at the Small Molecule MS Facility
(UCSD). Low-resolution MS measurements were made using a Thermoelectron Surveyor
UHPLC coupled to an MSD single-quadrupole detector. Preparative and analytical HPLC
was performed on an Agilent 1100 HPLC, or a Jasco system comprising dual-pumps (PU-
2086) and mixer, UV-vis detector (UV-2075) in tandem with an ELSD detector (Softa-A
model 300).
Animal Material. The sponge Trikentrion flabbeliforme (93-07-076) was collected
in Exmouth Gulf (800 m south of Bundegi Beach), Western Australia, in 1993 at a depth of
–
9 m using scuba. The sponge was an orange, pliable, ear-shaped specimen with a smooth
surface that, with pressure, emitted a red exudate. Microscopic spicule analysis revealed
the predominance of styles consistent with the species Trikentrion flabbeliforme. A
voucher sample of the sponge is archived at UC San Diego.
Extraction of Sponge and Isolation of (+)-trans-Herbindole A (14) and
Trikentramides E-I (14–18). Lyophilized Trikentrion flabbeliforme (63.88 g dry wt) was
extracted with CH Cl -MeOH (2 x 400 mL, 12 h), and the combined organic extracts were
2
2
concentrated and dissolved in H O-MeOH (1:9, 400 mL) prior to repeated extraction with
2
hexane (400 mL x 2). Concentration of the hexane-soluble layer gave Fraction A (1.4604
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1
1
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2
2
2
2
2
2
2
2
2
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3
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4
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5
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5
5
6
g). The aqueous-MeOH layer was adjusted to 2:3 H O-MeOH followed by extraction with
2
CH Cl (500 mL x 2) to yield Fraction B (0.6845 g). The aqueous layer was concentrated
2
2
and extracted with n-BuOH (400 mL x 2) to yield the C layer (0.8460 g). The remaining
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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9
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1
2
3
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8
9
0
1
2
3
4
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6
7
8
9
0
aqueous layer was dried to yield Fraction D (1.7995 g). Fraction B, the TLC of which
4
0
stained bright fuschia with vanillin-H SO , was separated further by size exclusion
2
4
chromatography (Sephadex LH-20, elution with MeOH) to give 10 fractions, which were
pooled according to TLC (10% MeOH-CH Cl ), UV-activity and staining with vanillin-
2
2
H SO in EtOH. Fractions 4 (0.0698 g) and 6 (0.0357 g) were further purified by reversed-
2
4
phase HPLC (Phenomenex, Kinetex C18 column, 150 x 21.2 mm, linear gradient, initial
conditions 75:25 H
CH
mg, 0.0074 % dry mass, t
= 18.21 min), G (16, 5.5 mg, 0.0086 % dry mass, t
dry mass, t = 15.12 mim) and I (18, 2.8 mg, 0.004 % dry mass, t
trikentramide E (14, 1.8 mg, 0.0028 % dry mass, t = 21.32 min) was obtained after
slightly different reversed-phase HPLC conditions (Phenomenex, Kinetex C18 column, 150
x 21.2 mm, linear gradient, initial conditions 75:25 H O-0.1% TFA-CH CN for three
minutes, 30:70 for 21 minutes to 100% CH CN for the last 2 minutes, flow rate = 12
2
O-0.1% TFA-CH
3
CN for three minutes, 30:70 for 17 minutes to 100%
–1
3
CN for the last 6 minutes, flow rate = 12 mL·min ) to give trans-herbindole A (13, 4.7
R
= 25.26 min), trikentramides F (15, 2.4 mg, 0.0038 % dry mass,
= 19.62 min), H (17, 1.6 mg, 0.0025
= 15.42 min). Pure
t
R
R
%
R
R
R
2
3
3
–
1
mL·min ).
(
+)-trans-Herbindole A (13): Gray oil; UV (CH CN) λmax 221 nm (logε 4.53), 271
3
2
3
(3.86); [α]D +29 (c 0.24, CHCl ); ECD (CH CN) λ 214 (Δε +5.3), 230 –7.3), 269 (–3.0),
3
3
–1
1
2
94 (+0.66); FTIR (ATR, ZnSe plate) ν 3430, 2953, 1393, 1375, and 727 cm ; H NMR
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+
1
3
and C NMR (CDCl ), Tables 1 and 2; HRESITOFMS m/z 214.1592 [M+H] (calcd for
3
+
C H N 214.1590).
15 20
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
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2
2
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0
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41
(
+)-Trikentramide E (14) : pale orange solid; UV (CH CN) λ 204 nm (logε
3 max
2
3
4
.10), 213 (4.08), 253 (3.63); [α]D +19 (c 0.18, CHCl ); ECD (CH CN) λ 215 (Δε –1.45);
3 3
–
1
1
FTIR (ATR, ZnSe plate) ν 3206, 2948, 2871, 1670 (s), 1626 (s), and 711 cm ; H NMR
+
1
3
and C NMR (CDCl ), Tables 1 and 2; HRESITOFMS m/z 230.1534 [M+H] (calcd for
3
+
C H NO 230.1539).
1
5
20
Trikentramide F (15): Dark yellow solid; UV (CH CN) λ 199 nm (logε 4.23),
3
max
2
3
218 (4.03), 245 (3.98), 326 (3.54); [α]D ~0 (c 0.12, CHCl ); ECD (CH CN) λ 210 (Δε
3
3
+
2.4), 214 (2.2), 252 (–0.7); FTIR (ATR, ZnSe plate) ν 3177, 2960, 2932, 1737 (s), 1726,
–
1
1
13
1
638, 1390 and 1377 cm ; H NMR and C NMR (CDCl ), Tables 1 and 2;
3
+
+
HRESITOFMS m/z 230.1173 [M+H] (calcd for C H NO 230.1176).
1
4
16
2
(
–)-Trikentramide G (16): Orange solid; UV (CH CN) λ 199 nm (logε 4.36), 218
3 max
2
3
(
4.14), 249 (4.14), 328 (3.70); [α]D –43 (c 0.55, CHCl ); ECD (CH CN) λ 192 (Δε –2.2),
3 3
2
03 (–2.6), 221 (+4.9), 255 (+1.1); FTIR (ATR, ZnSe plate) ν 3311 (br), 2946, 2872, 1738
–
1
1
13
(s), 1725 (s), 1626, 1592, 1382 and 1344 cm ; H NMR and C NMR (CDCl ), Tables 1
3
+
+
and 2; HRESITOFMS m/z 244.1331 [M+H] (calcd for C H NO 244.1332).
1
5
18
2
(+)-Trikentramide H (17): Pale yellow solid; UV (CH CN) λ 269 nm (logε 3.97),
3
max
23
2
21 (4.17); [α]D +22.6 (c 0.053, CHCl ); ECD (CH CN) λ 221 (Δε +8.6), 243 (–7.4), 272
3 3
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2
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6
(
+0.6); FTIR (ATR, ZnSe plate) ν 3311 (br), 2957, 2928, 1715 (s), 1704 (s), 1633 and ,
–
1
1
13
1
2
203 and 727 cm ; H NMR and C NMR (CDCl ), Tables 1 and 2; HRESITOFMS m/z
3
+
+
68.1309 [M+Na] (calcd for C H NO Na 268.1308).
1
5
19
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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0
1
2
3
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5
6
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8
9
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1
2
3
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0
(
+)-Trikentramide I (18): Tan solid; UV (CH CN) λ 221 nm (logε 3.96), 252
3 max
2
3
(3.39); [α]D +232 (c 0.093, CHCl ); ECD (CH CN) λ 224 (Δε –5.6), 243 (+5.0); FTIR
3 3
(ATR, ZnSe plate) ν 3397 (br), , 2958, 2925, 1681 (s), 1633 (s), 1447, 1204 1189, and
–
1
1
13
1138 cm ; H NMR and C NMR (CDCl ), Tables 1 and 2; HRESITOFMS m/z 268.1305
3
+
+
[M+Na] (calcd for C H NO Na 268.1308).
15 19 2
Reduction of 5-Methylisatin – Dioxindoles (S)-19 and (R)-19. Reduction of 5-
methylisatin (20, 200 mg, 1.2 mmol) with NaBH (was carried out according to the protocol
4
42
described by Hara and coworkers. The crude product was purified by silica flash
chromatography (1:1 EtOAc/CHCl ) to provide (±)-5-methylisatin [(±)-19] as a colorless
3
solid, 101 mg (50%). UV-vis (CH CN) λ 209 nm (logε 4.11), 248 (3.65), 296 (2.88).
3
max
+
1
13
LRMS m/z 164.20 [M+H] . The H and CNMR spectra of the product matched the
3
0
literature values for (R)-19.
A sample of (±)-19 (~100 µg) was resolved by chiral reverse-phased HPLC (Lux
U Amylose-2 column, 250 x 4.60 mm, isocratic conditions 2.5% i-PrOH-CH OH, flow
= 4.68 min) and (S)-19
5
3
–
1
rate = 1 mL·min ) to give the enantiomeric dioxindoles (R)-19 (t
R
(t
R
= 5.52 min). (S)-19: ECD (2.5% i-PrOH-CH
3
OH) λ 210 (Δε +54), 240 (–24.5), 267
30
(+7.4). (R)-19: λ 210 (Δε –54), 240 (+24.9), 267 (–7.0), [lit. 237 (–41), 264 (+12.5),
arbitrary units of ellipticity]. See Supporting Information for ECD spectra.
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Reduction of (–)-Trikentramide G (16). NaBH (1.40 mg, 35.8 µmol) was added
4
in portions to a solution of 16 (3.0 mg, 8.2 µmol) in anhydrous EtOH (1.0 mL) at room
temperature. After 1 hr., the solution was diluted with H O (2.0 mL), acidified to pH ~ 6
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
with HCl (1N), and extracted with CH Cl (2 x 2 mL). The organic layer was dried and the
2
2
residue (5.2 mg) was purified by reversed-phase HPLC (Phenomenex, Kinetex C18 column,
150 x 21.2 mm, linear gradient, initial conditions 75:25 H O-0.1% TFA-CH CN for three
CN for the last 6 minutes, flow rate = 12
= 15.13 min) and
= 15.43 min) with ECD and HPLC retention times identical with the
2
3
minutes, 30:70 for 17 minutes to 100% CH
3
–1
R R
mL·min ) to give 14 (0.5 mg, 18%, t = 19.30 min), 17 (1.0 mg, 33%, t
1
8, 1.2 mg, 40%, t
R
natural products.
Oxidation of (+)-trans-Herbindole A (13) to (–)-Trikentramide G (16).
(
+)-trans-Herbindole A (13) was oxidized according to the procedure of Wang and
3
3
coworkers. A solution of (+)-13 (1.0 mg, 4.7 µmol) in DMSO (0.05 mL) was treated with
a solution of iodine (1.4 mg, 5.6 µmol) and t-butyl hydroperoxide (70% w/v solution in
H
2
O, 3.0 µL, 23.4 µmol) in DMSO (0.50 mL) and heated to 80 ˚C with stirring for 18 h.
The solution was cooled, quenched with aqueous Na (5% w/v, 2.0 mL), extracted
with EtOAc (3 x 1.0 mL) and purified by reversed-phase HPLC (Luna C18 column, 250 x
.60 mm, linear gradient, initial conditions 65:35 H O-0.1% TFA-CH CN for three
minutes, 30:70 for 20 minutes to 100% CH CN for the last 6 minutes, flow rate = 1.0
2 2 3
S O
4
2
3
3
–
1
mL·min ) to give a product, as a yellow glass (0.5 mg, 44%) that was identical to natural
1
(
–)-trikentramide G (16) by MS, H NMR, ECD and HPLC retention time.
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ASSOCIATED CONTENT
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Supporting Information. CD spectra of (S)- and (R)-19, FTIR, H, C NMR and
2D NMR spectra of 13–18, DFT calculations of ent-6 and 13 (summaries and connection
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tables), H NMR simulation of the H-8 multiplet of 13 and molar rotations, [φ] , for 1-9,
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and 11-18 The Supporting Information is available free of charge on the ACS Publications
website at DOI: xxx
ACKNOWLEDGEMENTS
We thank S. Taylor and D. Manker for assistance with sponge collections, and X.
Cao (UCSD) for preliminary extractions and HPLC. We are grateful to S. Wang
(Soochow University, PRC) for helpful advice on oxidations of indoles to isatins, Y. Su
(UCSD) for HRMS data, and B. Duggan and A. Mrse (UCSD) for assistance with NMR
measurements. The 500 MHz NMR spectrometer and the HPLC TOFMS were purchased
with funding from the NSF (Chemical Research Instrument Fund, CHE0741968) and the
NIH Shared Instrument Grant (S10RR025636) programs, respectively. This work was
supported by a grant from NIH (R01 AI1007786).
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