The potential of achiral sponge-derived and synthetic bromoindoles as selective cytotoxins against PANC-1 tumor cells

Article abstract of DOI:10.1016/j.tet.2017.11.029

Our quest to isolate and characterize natural products with in vitro solid tumor selectivity is driven by access to repositories of Indo-Pacific sponge extracts. In this project an extract of a species of Haplosclerida sponge obtained from the US NCI Natural Products Repository displayed, by in vitro disk diffusion assay (DDA) and IC₅₀ determinations, selective cytotoxicity with modest potency to a human pancreatic cancer cell line (PANC-1) relative to the human lymphoblast leukemia cell line (CCRF-CEM). Two brominated indoles, the known 6-bromo conicamin (1) and the new derivative, 6-Br-8-keto-conicamin A (2), were identified and 2 (IC₅₀ 1.5 μM for the natural product vs 4.1 μM for the synthetic material) was determined to be responsible for the cytotoxic activity of the extract against the PANC-1 tumor cell line. The new natural product and ten additional analogs were prepared for further SAR testing.

Full text of DOI:10.1016/j.tet.2017.11.029

Tetrahedron xxx (2017) 1e7
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The potential of achiral sponge-derived and synthetic bromoindoles as
selective cytotoxins against PANC-1 tumor cells
Nicholas Lorig-Roach a, Frances Hamkins-Indik a, Tyler A. Johnson a, Karen Tenney a,
,
*
Frederick A. Valeriote b, Phillip Crews a
a Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, United States
b Department of Internal Medicine, Division of Hematology and Oncology, Henry Ford Hospital, Detroit, MI 48202, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Our quest to isolate and characterize natural products with in vitro solid tumor selectivity is driven by
access to repositories of Indo-Pacific sponge extracts. In this project an extract of a species of Haplo-
sclerida sponge obtained from the US NCI Natural Products Repository displayed, by in vitro disk
diffusion assay (DDA) and IC50 determinations, selective cytotoxicity with modest potency to a human
pancreatic cancer cell line (PANC-1) relative to the human lymphoblast leukemia cell line (CCRF-CEM).
Two brominated indoles, the known 6-bromo conicamin (1) and the new derivative, 6-Br-8-keto-con-
Received 23 October 2017
Received in revised form
9 November 2017
Accepted 10 November 2017
Available online xxx
icamin A (2), were identified and 2 (IC50 1.5 mM for the natural product vs 4.1 mM for the synthetic
material) was determined to be responsible for the cytotoxic activity of the extract against the PANC-1
tumor cell line. The new natural product and ten additional analogs were prepared for further SAR
testing.
Keywords:
PANC-1 cytotoxicity
Sponge-derived halogenated indoles
Synthetic halogenated indoles
© 2017 Elsevier Ltd. All rights reserved.
1. Introduction
hepatoma HepG2, and human brain U251N) compared to responses
from two normal cell lines (murine and human hematopoietic
We believe that the study of bioactive natural products from
sponges obtained within the Indonesian Coral Triangle, called by
some the “biodiversity bullseye,” represents an underexplored, rich
opportunity. One example of a significant result obtained from
exploration of the region is that manzamine A, a complex sponge-
derived antimalarial alkaloid1 (also a putative sponge-associated
bacterial product)2 and subject of sustained investigation,3 can be
isolated in large amounts from several Indonesian sponges. Moti-
vated by this outcome, we continue to evaluate sponge extracts
from repositories rich in Indonesian sponge taxa. Currently, these
resources consist of: 558 alcohol preserved sponge samples
collected by the UC Santa Cruz team, and 230 Indonesian marine
sponge extracts provided to UCSC from the NCI-DTP repository.4 An
important filter to prioritize work on these samples utilizes a pri-
mary in vitro disk diffusion assay (DDA) developed at the Henry
Ford Cancer Institute. This assay flags samples based on selectivity
and potency against five solid tumor cell lines (human pancreatic
cancer PANC-1, human ovarian OVCAR-5, human lung H125, human
progenitor cells, CFU-GM) and a leukemia cell line (CEM).5 The five
cancers represented by these cell lines account for 74% of all solid
tumor cancers (1,658,000 cases per year in the US) and 70% of solid
tumor mortality (589,000 deaths per year in the US).6 The output
from our primary screen is quantified by measuring the radius of
cell death after exposure to extract or compounds, where selec-
tivity is defined by a differential zone of inhibition between cell
lines.
The first phase of our effort to evaluate the sample library
described above has been to pinpoint extracts and compounds with
solid tumor selectivity in the DDA5 (those with a >6.5 mm differ-
ence in zone of inhibition for solid tumor cell lines vs. either bone
marrow progenitor or leukemia cells). During the last two years,
120 of the UCSC sponges along with 136 organic extracts of the NCI-
DTP sponge samples have been extracted, pre-fractionated, and
tested. The overall priority hit rate is low (y 1%), but there have
been a number of positive outcomes using this approach with
Indonesian sponges, including our recently published finding that
makaluvamine J can be isolated from both UCSC and DTP collec-
tions of Zyzzya fuliginosa. This compound exhibits solid tumor
selectivity for both PANC-1 (IC50
¼
54 nM) and OVCAR-5
* Corresponding author.
E-mail address: pcrews@ucsc.edu (P. Crews).
(IC50 ¼ 120 nM) cells.7 The general steps involved in the
https://doi.org/10.1016/j.tet.2017.11.029
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Please cite this article in press as: Lorig-Roach N, et al., The potential of achiral sponge-derived and synthetic bromoindoles as selective
cytotoxins against PANC-1 tumor cells, Tetrahedron (2017), https://doi.org/10.1016/j.tet.2017.11.029

2
N. Lorig-Roach et al. / Tetrahedron xxx (2017) 1e7
discovery process (Chart 1) have now been applied to the 256
samples in this assemblage and one chosen for detailed analysis has
provided the results reported herein, while outcomes from the
remaining sponge active extracts will be published elsewhere.
equivalent height, indicating the presence of a single bromine
atom. The two compounds briefly discussed above, known 6-Br-
conicamin (1)8 and new 6-Br-8-keto-conicamin (2), were separated
by semi-preparative reversed phase HPLC using 21:89 acetoni-
trile:H2O. Neither fraction formed adducts in ESI mass spectrom-
etry when spiked with sodium or potassium, consistent with a
native positive charge. Accurate mass measurement gave two
nearly identical molecular formulas differing by an oxygen: 1: ESI
m/z 279.0467 [M]þ (calcd for C13H16N729Br, 279.0491) and 2: ESI m/z
295.0419 [M]þ (calcd for C13H16N2O79Br, 295.0441). The mass data
from 1 coupled with a 1H NMR spectrum showing four indole ring
2. Results and discussion
The extract of Oceanapia sp. C011027 (from the NCI-DTP re-
pository) was prioritized for investigation in this study using the
soft agar disk diffusion assay (DDA) whose results are shown in
Table 1. The DDA measures a zone of inhibition (in mm) for a
sample diffusing from a disk placed at the edge of the petri dish
containing tumor cells embedded in a soft agar matrix. Samples
considered to have high cytotoxicity exhibit a zone of inhibition
ꢀ20 mm in any of the cell lines, which included five solid tumor cell
lines, a leukemia line (CEM), and bone marrow progenitor cells
(CFU-GM). Some benchmark results are provided by the activities
for two clinical drugs, gemcitabine and 5-fluorouracil, which are
powerful cytotoxins against almost all of the eight cell lines
screened. Both drugs also displayed selectivity between one or
more solid tumor cell lines and either the leukemia or a normal cell
surrogate (zone of inhibition difference > 7 mm, indicated by bold
numbers in Table 1). The best response for the Oceanapia sp. extract
was against PANC-1 (zone of inhibition ¼ 23 mm) which repre-
sented selective cytotoxicity relative to both CEM and murine CFU-
GM cells (Dinhibition zone ¼ 10 mm and 7 mm, respectively). These
results prompted creation of an HPLC peak library that was
screened in the DDA and the active fractions were further evaluated
by MSn and NMR. Subsequent isolation afforded the known Sub-
erites sponge compound, 6-Br-concamin (1)8 and an unreported
compound 6-Br-8-keto-concamin A (2) shown in Fig.1. While 1 was
not cytotoxic by the DDA, synthetic 2 (2a) at 112 nmol/disk
exhibited a zone of inhibition ¼ 28 mm against PANC-1 and 33 mm
against MCF-7, each with a greater than 20 mm differential be-
tween CEM and CFU-GM cell lines. We also synthesized a family of
bromoindole compounds bearing quaternary ammonium sub-
stituents including the 5-Br analog of 2 (3), which was also active in
the DDA with an inhibition zone of 20 mm against PANC-1 and
33 mm against MCF-7, again selective between these solid tumor
cell lines and CEM and CFU-GM. Eight other synthetic in-
termediates employed in the syntheses were inactive in the DDA.
Additional elements of the study included two comprehensive
protons, two vinyl protons and a singlet for nine protons near d3
allowed 1 to be identified as 6-bromo-conicamin (1) by der-
eplication. Despite the structural similarity implied by both MS and
NMR data, the structure of 2 had not been reported in the literature.
Inspection of the 13C NMR spectrum of 2 revealed that the vinyl
carbons in 1 were not present and were replaced by a ketone at dC
185.4 and a CH2 at dC 66.1, respectively (Table 1). The HMBC cor-
relations observed from the N-trimethyl protons at dH 3.33 to the
CH2 at dC 66.1 but not the carbonyl at dC 185.4 suggested a ketone-
methylene-trimethylamine motif. These data coupled with the
remaining long range correlations observed by gHMBCAD enabled
provisional assignment of 2. However, placement of the Br at either
C-5 or C-6 could not be confidently assigned. This ambiguity was
resolved by additional NMR
d analyses discussed next along with
NMR data comparison to synthesized products including 6-bromo-
8-keto-conicamin A (2a), and 5-bromo-8-keto-conicamin A (3).
Finalizing the Br atom placement in natural product 2 at C-5 vs.
C-6 (Fig. 1) should be trivial as it involves assigning the relative
position of an indole AB spin system as proximal or distant to the
NH. Unfortunately, our attempts to obtain NOE or HMBC correla-
tions to the NH residue for a definitive resolution were unsuc-
cessful. Alternatively, a literature search to provide NMR dH and dC
values distinctive for these isomeric frameworks provided diag-
nostic patterns shown in Fig. 1. First, the dH shifts at H-4/H-5 (3JH-H
portion), and H-7 for a 6-bromo isomer are different vs. those at H-
4, and H-6/H-7 (3JH-H portion) for a 5-bromo isomer as shown in
Fig. 2. A key is that the relative DdH for the o-proton doublets is
small (y0.2 ppm) for the 5-Br isomer but large (>0.5 ppm) for the
6-Br isomer. Second, the dC at C-3a is deshielded by approximately
3 ppm in the 5-Br relative to that of the 6-Br indole.9,10 The
observed patterns for 2 matched that of the lower example in Fig. 2:
vicinal proton DdH ¼ 0.69 and dC-3a ¼ 124.3. Correspondingly, the
synthetic compounds obtained as described in the next section
further supported these observations. For example, the spectrum of
3 matched that of the upper example in Fig. 2: the vicinal proton
evaluations: (a) unraveling NMR d values to facilitate the placement
of a halogen at the 5 vs. 6 position of an indole ring, and (b) using
the PANC-1 IC50's to define a useful SAR pattern for this indole
scaffold.
A DDA active fraction (data not shown herein) from the
Oceanapia peak library actually contained a mixture of two com-
pounds with m/z of 279 and 295, each having an Mþ2 peak of
DdH
¼
0.20 and dC-3a
¼
127.7 (see NMR data in Table 1,
Supplemental Figs. S9 & S10), and the data of the commercially
available aldehyde 5 matched that of the lower example in Fig. 2:
Chart 1. Overview of the campaign involving: sample acquisition, extraction, prefractionation, and Disk Diffusion Assay (DDA) for solid tumor selectivity against five human tumor
cell lines. The work flow also involved dereplication and structure elucidation by accurate MSn and NMR spectroscopy along with SAR biological evaluation of natural products and
synthetic analogs.
Please cite this article in press as: Lorig-Roach N, et al., The potential of achiral sponge-derived and synthetic bromoindoles as selective
cytotoxins against PANC-1 tumor cells, Tetrahedron (2017), https://doi.org/10.1016/j.tet.2017.11.029

N. Lorig-Roach et al. / Tetrahedron xxx (2017) 1e7
3
Table 1
Disk diffusion assay zone of inhibition data for the parent Oceanapia sp. C011027 extract, synthetic 2a, various indole analogs of 2, and therapeutic standards compared across
five solid tumor cell lines, a leukemia line (CEM), and bone marrow progenitor cells (CFU-GM). Bold numbers indicate selectivity, defined as a differential zone of inhibition
greater than 7 mm between solid tumor cell line and at least one normal cell proxy (CEM/CFU-GM).
Table 2
NMR Dataa (DMSO-d6) for natural 6-Br-8-keto-conicamin A (2), synthetic 6-Br-8-keto-conicamin (2a), and synthetic 5-Br-8-keto-conicamin A (3).
Position
2
2a
3
dC, Typeb
dH, mult (J, Hz)
HMBCc
dC
dH, mult (J, Hz)
dC
dH, mult (J, Hz)
1
8.47, bs
8.51, s
8.54, s
2
3
3a
4
5
6
7
7a
136.9, CH
114.6, C
124.3, C
122.7, CH
125.4, CH
115.9, C
115.6, CH
138.1, C
8.58, bs
e
3, 3a, 7a
138.2
114.5
124.8
122.5
125.0
115.5
116.1
139.5
184.6
66.1
8.51, s
e
138.8
114.1
127.7
123.0
115.6
125.4
114.8
137.6
184.4
66.0
8.52, s
e
e
e
e
8.08, d (8.5)
7.39, dd (1.3, 8.5)
3, 3a, 5, 7, 7a
3a, 7
8.07, d (8.5)
7.35, bd (8.5)
8.27, s
e
e
e
7.34, d (8.4)
7.54, d (8.6)
e
7.77, bs
e
3a, 5, 6, 7a
7.78, bs
e
8
9
185.4, C
66.1, CH2
53.5, CH3
e
e
e
4.99, s
3.33, s
8, N-(CH3)3
9
4.90, s
3.31, s
4.91, s
3.32, s
N-(CH3)3
53.5
53.5
a
Measured at 600 MHz (1H) and 150 MHz (13C).
Carbon type determined by gHSQC.
gHMBC(AD) correlations to indicated carbon with JnCH optimized for 6 Hz couplings and using a 2 s relaxation delay.
b
c
the vicinal proton DdH ¼ 0.67 and dC-3a ¼ 123.1.
The synthetic work to prepare 2 and its analogs provided
additional information to probe the structure-activity relationship
describing the cytotoxicity of Br-indoles. Based on our DDA and IC50
screening of the intermediates and products from the synthesis,
only the compounds most similar to 2 (such as 3 and 13) possessed
notable cytotoxic activity as shown in Tables 1 and 3. The DDA data
(Table 1) indicates similar solid tumor selectivity for 2 and 3 in
The process to obtain compound 2 and other analogs by syn-
thesis is shown in Scheme 1. Two indole-3-carboxaldehydes, 4 and
5, were used as separate starting materials to provide the oxo-
tryptamine synthons 10 and 11. While the yield of the cyanohydrin
silylether 6 from 4 match that in the literature (60%), the yield of 7
from 5 was poor (20%) vs that in the literature for the 5-Br analog
(88%).11 Oxidation of both 6 and 7 via DDQ provided high yields of 8
(83%) and 9 (60%).11 Hydrogenation of 8 afforded 10 in good yield
after workup (76%) and reduction of 9 provided the first route to
11.12 Bromination of the keto-tryptamine synthon 10 proceeded in a
straightforward fashion, producing the bromo-keto-tryptamines 11
and 12 which were separated by reversed phase HPLC.13 We used
11 prepared from 5 to confidently distinguish between the 5- and
6-bromo isomers (11 and 12) prepared via bromination of 10. NMR
spectroscopic experiments on the commercially obtained 5 assured
correct assignment (see SI Figs. S34-38). The final steps involved
methylation of 10, 11, or 12 to respectively afford 13, 2a, or 3. Per-
forming these reactions with a reduced molar ratio of methyl iodide
provided the dimethyl analogs 14, 15, or 16 from 11, 12, or 10,
respectively as minor products. The compounds 2a, 11, or 14 were
produced in duplicate from either 5 or 10 as starting material and
were equivalent in their spectroscopic and bioassay properties.
Fig. 1. The major products, 1 and 2, isolated from Oceanapia sp. C011027 and the 5-Br
synthetic analog 3.
Please cite this article in press as: Lorig-Roach N, et al., The potential of achiral sponge-derived and synthetic bromoindoles as selective
cytotoxins against PANC-1 tumor cells, Tetrahedron (2017), https://doi.org/10.1016/j.tet.2017.11.029

4
N. Lorig-Roach et al. / Tetrahedron xxx (2017) 1e7
Fig. 2. Using NMR data to rapidly distinguish between 5- vs. 6-Br indole frameworks when the diagnostic NOEs to the NH cannot be observed. a This work; b Seagraves;10 cBartik.9
Scheme 1. Synthesis of the natural product 6-Br-8-keto-conicamin (2a) and nine key analogs 3, 8, and 10e16 for in vitro cytotoxicity screening against the PANC-1 tumor cell line.
Following the synthetic route to 11 from 5 enabled facile differentiation between the 5- and 6-bromo isomers 11 and 12 synthesized from 10. The spectral and biological properties
of 2a, 11, or 14 derived from 5 were equivalent to those derived from 10.
comparison to the two therapeutic standards. The IC50 data
(Table 3) reveals that a quaternary amine functionality is vital for
low mM activity against PANC-1 cells, which is on a par with that of
Also, the presence of the Br atom at C-5 or C-6 on the indole ring
and the keto group at C-8 are significant factors in the IC50 activity
of 2 and 3. Interestingly, relative to 2, the primary amines (com-
pound 10e12) and tertiary amines (compounds 14e16) were
inactive in both the DDA and the IC50 determinations. In addition,
13, which lacks a Br, was not selective in the DDA screen and was an
order of magnitude less potent vs. 2 against PANC-1 cells based on
IC50 data. While the position of the Br (at C-5 or C-6) is less
important, it was the 6-bromo-8-keto-conicamin (2) that exhibited
the best DDA selectivity for PANC-1 and MCF-7, and greatest po-
tency against PANC-1 (IC50 data against MCF-7 were not measured).
Overall, the low micromolar in vitro activity of 2 against the PANC-
the two therapeutic standards, 5-flourouracil and gemcitabine.
Table 3
In vitro IC50 values against the PANC-1 cell line for natural 2, synthetic 2a, other
synthetic analogs, and two therapeutic standards.
Compound
PANC-1
IC50 (mM)
6-Br-conicamin (1)
NA
1.5
4.1
5.8
NA
NA
NA
NA
23
6-Br-8-keto-conicamin A (2) [natural product]
6-Br-8-keto-conicamin A (2a) [synthetic product]
5-Br-8-keto-conicamin A (3)
1H-indole-3-carbonyl cyanide (8)
8-keto-tryptamine (10)
1 cell line (IC50 1.5
mM for the natural product vs 4.1 mM for the
synthetic material) is exciting and is similar to that of the clinical
therapeutics (5FU: IC50 ¼ 7.0
mM; gemcitabine: IC50 ¼ 0.02
mM).
6-Br-8-keto-tryptamine (11)
5-Br-8-keto-tryptamine (12)
3. Conclusion
N,N,N-trimethyl 8-keto-tryptamine (13)
6-Br-N,N-dimethyl-8-keto-tryptamine (14)
5-Br-N,N-dimethyl-8-keto-tryptamine (15)
N,N-dimethyl-8-keto-tryptamine (16)
5-fluorouracil
NA
NA
NA
The halogenated indole moiety found in 2 has been observed in
a number of metabolites from marine macro- and microorganisms,
including the topsentins,9 aplysinopsins,10 dragmacidins,16 hypa-
phorines,17 didemnimides,18 and nakijinamines.8 Thorough reviews
of marine-derived indole alkaloids and their activities has been
published elsewhere.19,20 The bioactivity of these compounds is
diverse: cytotoxicity, antibacterial activity, anti-fungal activity,
5.714; 7.0
gemcitabine
7.215; 0.02a
NA e compounds inactive in soft-agar disk diffusion assay as shown in Table 1.
a
Note that this IC50 was determined after 5 days compared to 48 h. We have
found in our lab (unpublished data) that there is ~250-fold difference in IC50 values
between a 24 h and a 5-day assay for gemcitabine.
Please cite this article in press as: Lorig-Roach N, et al., The potential of achiral sponge-derived and synthetic bromoindoles as selective
cytotoxins against PANC-1 tumor cells, Tetrahedron (2017), https://doi.org/10.1016/j.tet.2017.11.029

N. Lorig-Roach et al. / Tetrahedron xxx (2017) 1e7
5
histamine receptor agonism, and neurotransmitter receptor affin-
ity. The quaternary amine functionality found in 2 is rare, but there
are examples of bioactive natural products containing this cationic
residue attached to a halogenated indole scaffold (Fig. 3). A pre-
nylated indole from a bryozoan with moderate nAChR activity
prompted medicinal chemistry efforts in which some analogs (e.g.
18) attained high nanomolar affinity for a variety of nAChR sub-
types.21 The results of this study are modest, yet the in vitro cyto-
toxicity of 2 against the PANC-1 cell line exceeds that reported for
5-FU and gemcitabine, which are the primary constituents of the
current standards of care for pancreatic cancer.14,15 The structure of
2 was confirmed by synthesis and a number of analogs were made
using methodology originally developed for the syntheses of the
rhopaladins, dragmacidins, and almazoles.11e13 The quaternary
amine functionality and bromination of the indole ring of 2 was
critical for activity against PANC-1. The new small molecule
discovered in this work highlights the value of continued pursuit of
achiral marine natural products and the effectiveness of continued
screening using the NCI Developmental Therapeutics Program
extract library. Overall, the lack of small molecule therapies to treat
pancreatic cancer makes the results presented in Table 3 reasonably
significant.
evaporative light scattering detector (ELSD). Optical rotations
were performed on a JASCO P-2000 polarimeter; the average of
10 measurements is reported, each with 10 s digital integration
time and 12 s cycle time. All NMR experiments were run on a
Varian Unity spectrometer (500 and 125 MHz for 1H and 13C,
respectively) or Varian INOVA spectrometer (600 and 150 MHz
for 1H and 13C, respectively) equipped with a cryoprobe. High
accuracy mass spectrometry measurements were recorded using
an Applied Biosystems Mariner 5200 electrospray ionization
time of flight (ESI-TOF) mass spectrometer. Synthetic procedures
were either performed via Schlenk line with nitrogen or via
balloon and septa techniques with argon. Flash chromatography
was performed using a Biotage Isolera Prime fraction collector
with a 100 g silica gel 60 column.
4.2. Biological material
The sponge Oceanapia sp. C011027 (phylum Porifera, class
Demospongia, order Haplosclerida) was collected at 10 m depth off
the coast of Sulawesi, Indonesia. Sample collection and identifica-
tion was done by the Coral Reef Foundation under contract to the
National Cancer Institute (National Institutes of Health). A taxo-
nomic voucher specimen (number 0CDN1333) was deposited at the
Smithsonian Institution in Suitland, MD (USA).
4. Experimental
4.3. Compound isolation
4.1. General experimental procedures
After extract C011027 demonstrated solid tumor selective
activity, an HPLC peak library was generated to determine the
UV spectra were measured with Thermo Ultimate 3000 DAD.
IR spectra were measured with a Perkin-Elmer Spectrum One
FTIR spectrometer. Analytical LC-MS analysis was performed on
samples at a concentration of 10 mg/mL for crude extracts and
active constituents. The extract was fractionated using
a
250 ꢁ 10 mm Phenomonex Luna C18 5
m
m semi-preparative
column equipped with an analytical guard column
(4.0 ꢁ 3.0 mm C18 cartridge) and two Waters 515 HPLC pumps.
The eluent was monitored by a Waters 996 detector before being
fractionated by a Gilson 215 Liquid Handler that generated one
1 mg/mL for pure compounds, using a 150 ꢁ 4.60 mm 5
Phenomenex Luna column in conjunction with a guard column
L vol-
mm C18
and 4.0 ꢁ 3.0 mm C18 cartridge (Phenomenex, Inc.). 20
m
umes of sample were injected onto the column using Waters 717
autosampler, with a flow rate of 1 mL/min provided by two
Waters 515 HPLC pumps. The eluent was split between an
Applied Biosystems Mariner 5200 electrospray ionization time-
of-flight (ESI-TOF) mass spectrometer and a Waters 996 photo-
fraction per minute. A Waters 717 autosampler injected 100 mL of
extract at 70 mg/mL onto the column; the first gradient 10:90
acetonitrile:H2O was held for 1 min, then linearly shifted to
100:0 acetonitrile:H2O over 29 min, and finally held at 100:0
acetonitrile:H2O for 10 min. Fractions H16 and H17 were the only
fractions with significant zones of inhibition when evaluated in
the DDA. These contained a mixture of 1 and 2. Compounds 1 and
2 were separated using the following isocratic conditions: 21:79
acetonitrile:H2O with tR ¼ 12.5 and 15.5, respectively. Note that
the charge of these compounds leads to significant tailing and
inconsistent retention times over longer timescales (i.e. weeks).
This can be mitigated to some extent by using TFA in the mobile
phase. Fraction collection was done manually using the same
column as above with two Waters 510 HPLC pumps, a Waters
Model 680 gradient controller, and an ABI Analytical Spectroflow
783 UV detector monitoring at 230 nm.
diode array (PDA) UV detector in line with
a SEDERE 75
4.3.1. 6-Bromo-conicamin (1)
HAESIMS m/z 279.04667 [M]þ (calcd for C13H16N729Br,
279.04914). NMR spectra were in accordance with literature.8
4.3.2. 6-Bromo-8-keto-conicamin (2)
Light pink crystalline solid; UV (MeCN:H2O) 209, 247, 271,
308 nm; IR (film) ymax 3438, 3250, 1637, 1520, 1443, 1414, 1384,
1048, 891, 595 cmꢂ1; 1H NMR (DMSO-d6, 600 MHz)
d 8.58 (1H, bs,
H-2), 8.08 (1H, d, J ¼ 8.5 Hz, H-4), 7.77 (1H, s, H-7), 7.39 (1H, dd,
J ¼ 1.3, 8.5 Hz, H-5), 4.99 (2H, s, H-10), 3.33 (9H, s, N-(CH3)3); 13C
NMR (DMSO-d6, 150 MHz)
d
185.4 (C, C-20), 138.1 (C, C-7a), 136.9
(CH, C-2), 125.4 (CH, C-5), 124.3 (C, C-3a), 122.7 (CH, C-4), 115.9 (C,
Fig. 3. Other natural products featuring halogenated indole with a quaternary amine
moiety.
C-6), 115.6 (CH, C-7), 114.6 (C, C-3), 66.1 (CH2, C-10), 53.5 (CH3, N-
Please cite this article in press as: Lorig-Roach N, et al., The potential of achiral sponge-derived and synthetic bromoindoles as selective
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6
N. Lorig-Roach et al. / Tetrahedron xxx (2017) 1e7
(CH3)3); HAESIMS m/z 295.04191 [M]þ (calcd for C13H16N2O79Br,
4.3.9. 5-Bromo-8-keto-tryptamine (12)
295.04405).
The second procedure above for 11 also afforded 12 by reversed
phase HPLC. 1H NMR (DMSO, 500 MHz)
d
8.54 (1H, d J ¼ 3.0 Hz, H-
2), 8.29 (1H, d J ¼ 1.8 Hz, H-4), 7.53 (1H, d J ¼ 8.6 Hz, H-7), 7.41 (1H,
4.3.3. 2-(1H-indole-3-yl)-2-(trimethylsiloxy)acetonitrile (6)
dd J ¼ 8.6, 1.7 Hz, H-6), 4.39 (2H, s, H-9); 13C NMR (DMSO, 125 MHz)
Procedure adapted from Janosik.11 To a solution of 1.16 g 1H-
indol-3-yl-carboxaldehyde (Combi-Blocks, San Diego, CA, USA) in
10 mL acetonitrile, 1.3 mL TMSCN was added. The reaction solution
was heated and maintained at reflux for 2 h before cooling. After
evaporation of the solvent, reaction products were purified from
the resulting oil using flash chromatography (isocratic 80:20
hexane-ethyl acetate) to afford 6 (1.2 g, 65%) as a lime-green oil. MS
(ESI) 243 m/z (MꢂH)-. NMR data were in accordance with literature.
d
187, 136.3, 135.4, 126.8, 125.9, 123.0, 115.1, 114.7, 112.6, 44.1. MS
(ESI) 252/254 [MþH]þ.
4.3.10. 8-Keto-N,N,N-trimethyltryptamine (13)
To a solution of 8-keto-tryptamine (10) in KHCO3-saturated
MeOH, 3 equivalents of methyl iodide were added and stirred
overnight. The product mixture under these conditions contained
unreacted starting material (10), 13, 14, and a minor amount of the
single methyl analog which were separated by reverse phase HPLC.
Using excess MeI (>5 eq) yields exclusively the quaternary amine
4.3.4. 2-(6-Bromo-1H-indol-3-yl)-2-(trimethylsiloxy)acetonitrile
(7)
product. Yellow solid; 1H NMR (DMSO 600 MHz)
(1H, d J ¼ 7.3 Hz), 7.57 (1H, d J ¼ 7.3 Hz), 7.24 (2H, m), 4.93 (2H, s),
3.32 (9H, s). 13C NMR (DMSO, 150 MHz)
184.9, 137.4, 136.5, 125.4,
d 8.51 (1H, s), 8.15
Adapted from procedure for 5-bromo isomer from Janosik.11 To a
solution of 1.08 g 6-bromo-1H-indol-3-yl-carboxaldehyde (Tokyo
Chemical Industry, Tokyo, Japan) in 10 mL DME, 0.73 mL TMSCN
was added. The reaction solution was heated and maintained at
reflux for 2.5 h before cooling. After evaporation of the solvent,
reaction products were purified by flash chromatography (hexane-
ethyl acetate gradient) to afford 7 (0.35 g, 19%) as a light yellow oil.
d
123.3, 122.4, 121.0, 114.6, 113.1, 66.05, 53.5. HAESIMS m/z 217.1327
[MþH]þ (cald for C13H17ON2, 217.1341).
4.3.11. 6-Bromo-8-keto-conicamin (2a)
1H NMR (DMSO-d6, 600 MHz)
d
11.45 (1H, S), 7.63 (1H, d, J ¼ 1.9),
See above procedure for 13, but using compound 11 as starting
material. The product mixture primarily contained 2a and 14,
which were seprable by reverse phase HPLC. Off-white crystalline
7.60 (1H, d, J ¼ 8.4), 7.56 (1H, d, J ¼ 2.6), 7.24 (1H, dd, J ¼ 8.5, 1.8),
6.18 (1H, s), ꢂ0.11 (9H, s); 13C NMR (DMSO-d6, 150 MHz)
d 137.4,
solid; 1H NMR (DMSO 600 MHz)
d
8.51 (1H, s), 8.07 (1H,
126.0, 123.8, 122.5, 120.4, 119.9, 114.7, 114.6, 111.0, 56.8, ꢂ0.23. MS
(ESI) 321 and 323 m/z (MꢂH)ꢂ.
d J ¼ 8.41 Hz), 7.78 (1H, s), 7.35 (1H, d J ¼ 8.46 Hz), 4.90 (2H s), 3.31
(9H, s). 13C NMR (DMSO,150 MHz)
d 184.6, 139.5, 138.2,125.0,124.8,
122.5, 116.1, 115.5, 114.5, 66.1, 53.5. HAESIMS m/z 295.0443 [M]þ
(calcd for C13H16N2O79Br, 295.0441).
4.3.5. 1H-indol-3-carbonyl cyanide (8)
A solution of DDQ (1.21 g, 5.3 mmol) in 60 mL dioxane was
added dropwise by addition funnel to a stirring solution of 6 (1.18 g,
4.8 mmol) in 10 mL dioxane. After 2.5 h the reaction mixture was
filtered and the filtrate dried to a purple-black solid. Flash chro-
matography (DCM-EtOAc gradient) afforded 8 (0.68 g, 83%) as a
yellow oil. MS (ESI) 171 m/z [MþH]þ. NMR data were in accordance
with literature.
4.3.12. 5-Bromo-8-keto-conicamin (3)
See above procedure for 13, but using compound 12 as starting
material. The product mixture primarily contained 3 and 15. Off-
white crystalline solid; 1H NMR (DMSO 600 MHz)
d
8.52 (1H, s),
8.27 (1H, s), 7.54 (1H, d, J ¼ 8.3 Hz), 7.34 (1H, d, J ¼ 8.0), 4.91 (2H, s),
3.32 (9H, s). 13C NMR (DMSO, 150 MHz)
184.4, 138.8, 137.6, 127.7,
d
125.4, 123.0, 115.6, 114.8, 114.0, 66.0, 53.5. HAESIMS m/z 295.0442
[M]þ (calcd for C13H16N2O79Br, 295.0441).
4.3.6. 6-Bromo-1H-indol-3-carbonyl cyanide (9)
The procedure above was used with 6-bromo-indole-TMS (7)
(0.35 g, 1.1 mmol) and DDQ (0.27 g). Flash chromatography (hex-
anes-EtOAc gradient) afforded 9 (0.15 g, 55%) as a fluffy yellow
solid. MS (ESI) 247/249 [MꢂH]ꢂ.
4.3.13. 6-Bromo-8-keto-N,N-dimethyltryptamine (14)
See above procedure for 13, but using compound 11 as starting
material. 1H NMR (DMSO 600 MHz)
d12.26 (1H, s), 8.45 (1H, s), 8.10
(1H, d, J ¼ 8.5 Hz), 7.68 (1H, s), 7.31 (1H, dd, J ¼ 8.4, 1.4 Hz), 3.52 (2H,
s), 2.26 (6H, s) 13C NMR (DMSO, 150 MHz) see Supplemental
Figs. S29 and S30. HAESIMS m/z 281.0285 [MþH]þ (calcd for
4.3.7. 8-Keto-tryptamine (10)
To 150 mg of 10% Pd/C, a solution of 1 mmol of 1H-indol-3-
carbonyl cyanide (8) in 10 mL acetic acid was added. Hydrogen
gas was added via balloon and septum. After 15 h, the reaction
mixture was filtered using diatomaceous earth and the filtrate was
dried, resuspended in water, basified, then extracted with ethyl
acetate to give 0.76 mmol of the free base 10. NMR data were in
accordance with literature.
C
12H14N2O79Br, 281.0290).
4.3.14. 5-Bromo-8-keto-N,N-dimethyltryptamine (15)
See above procedure for 13, but using compound 12 as starting
material. 1H NMR (DMSO 600 MHz)
d8.46 (1H, s), 8.30 (1H, bs), 7.46
(1H, d, J ¼ 8.6 Hz), 7.32 (1H, broad d, J ¼ 8.6 Hz), 3.51 (2H, s), 2.26
(6H, s) 13C NMR (DMSO, 150 MHz) see Supplemental Figs. S32 and
S33. HAESIMS m/z 281.0287 [MþH]þ (calcd for C12H14N2O79Br,
281.0290).
4.3.8. 6-Bromo-8-keto-tryptamine (11)
The first route to 11 employed the procedure above (10) using 9
as starting material. The second route was performed as follows. To
a stirring solution of
b
-keto-tryptamine (10) in 1:1 formic acid:-
4.3.15. 8-oxo-N,N-dimethyltryptamine (16)
acetic acid, 1.2 equivalents of Br2 was added dropwise. After 24 h,
the reaction mixture was dried and subjected to reversed phase
See above procedure for 13. 1H NMR (DMSO 600 MHz)
d 11.98
(1H, s), 8.42 (1H, d, J ¼ 2.7), 8.17 (1H, d, J ¼ 7.9 Hz), 8.15 (1H,
d J ¼ 7.29 Hz), 7.47 (1H, d, J ¼ 7.7 Hz), 7.20 (1H, td, J ¼ 7.7, 7.1, 1.5 Hz),
7.17 (1H, td, J ¼ 7.9, 7.0, 1.3 Hz), 3.52 (2H, s), 2.27 (6H, s). 13C NMR via
HPLC to afford 11 and 12. 1H NMR (DMSO, 500 MHz)
d 12.47 (1H, s,
NH), 8.52 (1H, d, J ¼ 3 Hz, H-2), 8.1 (1H, d J ¼ 8.4 Hz, H-4), 7.74 (1H, s,
H-7), 7.4 (1H, d J ¼ 8.5 Hz, H-5), 4.38 (2H, s, H-9); 13C NMR (DMSO,
HMBC (DMSO, 150 MHz)
d 192.9, 136.2, 135.9, 125.6, 122.6, 121.6,
125 MHz)
d
187 (CO), 137.5, 136, 123.3, 124.1, 122.6, 115.6, 115.8,
121.3, 115.0, 112.1, 66.4, 45.4. HAESIMS m/z 203.1179 [MþH]þ (cald
115.2, 113.1, 44.1. MS (ESI) 252/254 [MþH]þ.
for C12H15ON2, 203.1184).
Please cite this article in press as: Lorig-Roach N, et al., The potential of achiral sponge-derived and synthetic bromoindoles as selective
cytotoxins against PANC-1 tumor cells, Tetrahedron (2017), https://doi.org/10.1016/j.tet.2017.11.029

N. Lorig-Roach et al. / Tetrahedron xxx (2017) 1e7
7
4.4. Cytotoxic assays
Acknowledgments
Extract C011027 was evaluated in the in vitro disk diffusion soft
agar colony formation assay which defines differential cell killing
among 13 cell types. The screen includes CCRF-CEM (leukemia),
H125 (lung cancer), OVCAR-5 (ovarian cancer), MCF-7 (breast
cancer), PANC-1 (pancreatic cancer), U251N (brain cancer), as well
as a normal murine hematopoietic progenitor cell, CFU-GM. The
human cancer cell lines are maintained in cell culture in either
Dulbecco's modified MEM, Eagles MEM or RPMI-1640 depending
upon the cell lines. The media is supplemented with either 10% or
We thank the Natural Products Branch, Developmental Thera-
peutics Program, Division of Cancer Treatment and Diagnosis, Na-
tional Cancer Institute for collection and extraction of Oceanapia
sp.27. This work was supported by a grant from the NIH R01
CA47135 (P.C. and F.A.V.). We also acknowledge funding from NSF
CHE1427922 for the Thermo Velos Pro electrospray ionization
hybrid ion trap-orbitrap mass spectrometer and the NIH
1S10OD018455-01 for the 800 MHz NMR spectrometer and helium
cryoprobe. Finally, we thank the members of the Lokey research
group at UCSC, especially M. Naylor and J. Schwochert, for their
kind advice during the synthetic portion of this work.
15% BCS plus Pen-Strept (100,000 I.U. Pen/L; 100,000
mg/L Strept)
and -glutamine (292 mg/L). Cells are removed from cultures by a
L
trypsin solution (0.05%). Plating efficiencies are sufficiently high
that 30,000 to 60,000 cells in 3 mL produce the desired number of
colonies (over 10,000 per plate) in 60 mm plates. This soft agar top
layer (0.3% with the serum and media as above) plus the titrated
tumor cells are poured into plates and allowed to solidify. A volume
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.tet.2017.11.029.
of 15 mL of each sample is dropped onto a 6.5 mm disk (Whatman
filter disks). The disk is allowed to dry overnight and then placed
close to the edge of the petri dish. The plates are incubated for 7e10
days (depending upon cell type) and examined by an inverted
stereo-microscope (10ꢁ) for measurement of the zone of inhibition
measured from the edge of the filter disk to the beginning of
normal-sized colony formation. The diameter of the filter disk,
6.5 mm, and a difference in zones between solid tumor cells and
either normal or leukemia cells of 6.5 mm or greater defines solid
tumor selective compounds. If the test material is excessively toxic
at the first dosage, a range of dilutions of the agents (at 1:4 dec-
rements) are tested against the same tumors. At some dilution,
quantifiable cytotoxicity is invariably obtained.
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Please cite this article in press as: Lorig-Roach N, et al., The potential of achiral sponge-derived and synthetic bromoindoles as selective
cytotoxins against PANC-1 tumor cells, Tetrahedron (2017), https://doi.org/10.1016/j.tet.2017.11.029