Synthesis and biological evaluation of isomeric methoxy substitutions on anti-cancer indolyl-pyridinyl-propenones: Effects on potency and mode of activity

Article abstract of DOI:10.1016/j.ejmech.2016.06.016

Certain indolyl-pyridinyl-propenone analogues kill glioblastoma cells that have become resistant to conventional therapeutic drugs. Some of these analogues induce a novel form of non-apoptotic cell death called methuosis, while others primarily cause micr

Full text of DOI:10.1016/j.ejmech.2016.06.016

Accepted Manuscript
Synthesis and biological evaluation of isomeric methoxy substitutions on anti-cancer
indolyl-pyridinyl-propenones: Effects on potency and mode of activity
Christopher J. Trabbic, Sage M. George, Evan M. Alexander, Shengnan Du, Jennifer
M. Offenbacher, Emily J. Crissman, Jean H. Overmeyer, William A. Maltese, Paul W.
Erhardt
PII:
S0223-5234(16)30494-9
10.1016/j.ejmech.2016.06.016
EJMECH 8677
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 April 2016
Revised Date: 7 June 2016
Accepted Date: 10 June 2016
Please cite this article as: C.J. Trabbic, S.M. George, E.M. Alexander, S. Du, J.M. Offenbacher, E.J.
Crissman, J.H. Overmeyer, W.A. Maltese, P.W. Erhardt, Synthesis and biological evaluation of isomeric
methoxy substitutions on anti-cancer indolyl-pyridinyl-propenones: Effects on potency and mode of
activity, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.06.016.
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Glioblastoma Cells
(red - tubulin; blue - nuclei)
Microtubule Disruption
Methuosis

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Synthesis and Biological Evaluation of Isomeric Methoxy Substitutions
on Anti-Cancer Indolyl-Pyridinyl-Propenones: Effects on Potency and
Mode of Activity
Christopher J. Trabbic a, Sage M. George b, Evan M. Alexander a,1, Shengnan Du b, Jennifer M.
Offenbacher a, Emily J. Crissman a, Jean H. Overmeyer b, William A. Maltese b,*, Paul W.
Erhardt a,*
aCenter for Drug Design and Development, Department of Medicinal and Biological Chemistry,
University of Toledo College of Pharmacy and Pharmaceutical Sciences, 2801 W. Bancroft
Ave., Toledo, OH 43606
bDepartment of Biochemistry and Cancer Biology, University of Toledo College of Medicine
and Life Sciences, 3000 Arlington Ave., Toledo, OH 43614
1 Present address: Department of Medicinal Chemistry, University of Minnesota College of
Pharmacy, 8-101 Weaver Densford Hall, 308 Harvard St. S.E., Minneapolis, MN 55455.
*Corresponding authors.
Email addresses: william.maltese@utoledo.edu (W. A. Maltese) and
paul.erhardt@utoledo.edu (P.W. Erhardt).
KEYWORDS. Methuosis, indolyl-pyridinyl-propenones, glioblastoma, microtubule disruption, cell death
ABSTRACT
Certain indolyl-pyridinyl-propenone analogues kill glioblastoma cells that have become
resistant to conventional therapeutic drugs. Some of these analogues induce a novel form of
non-apoptotic cell death called methuosis, while others primarily cause microtubule disruption.
Ready access to 5-indole substitution has allowed characterization of this position to be
important for both types of mechanisms when a simple methoxy group is present. We now
report the syntheses and biological effects of isomeric methoxy substitutions on the indole
ring. Additionally, analogues containing a trimethoxyphenyl group in place of the pyridinyl
moiety were evaluated for anticancer activity. The results demonstrate that the location of the
methoxy group can alter both the potency and the mechanism of cell death. Remarkably,
changing the methoxy from the 5-position to the 6-position switched the biological activity from

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induction of methuosis to disruption of microtubules. The latter may therefore represent a
prototype for a new class of mitotic inhibitors with potential therapeutic utility.
1. Introduction
Glioblastoma multiforme (GM) remains a lethal cancer due to rapid progression and
limited treatment options, namely surgical removal of the tumor followed by combined
radiotherapy and chemotherapy with temozolomide [1,2]. Recurrence of disease is usually
untreatable as a result of acquired drug-resistance and invasive dissemination of the tumor.
Temozolomide relies on triggering programmed cell death via activation of apoptosis [3].
However, GM cells harbor specific mutations in genes that are required to promote an efficient
apoptotic response [4,5]. Stimulation of nonconventional cell death pathways offers a possible
solution for treating drug-resistant cancers that are able to circumvent apoptosis [6,7].
Methuosis is a recently identified caspase-independent form of cell death that displays
characteristics distinct from other types of non-apoptotic cell death, such as necroptosis or
autophagy [8,9]. In cultured glioblastoma cells, methuosis begins with defective
macropinocytotic trafficking, causing the formation of large fluid-filled vacuoles. Accumulation
of vacuoles ultimately displaces the cytoplasm and the cell membrane loses integrity and
ruptures. While dysfunctional vesicular trafficking and accumulation of vacuoles appear to
contribute to cell death, there is evidence that additional metabolic or cellular insults are
required for execution of the methuosis cell death program [8,10,11].
The methuosis phenotype was initially observed by the ectopic expression of activated
Ras and Rac GTPases in GM cells [12,13]. More recent studies have focused on the pursuit of
small molecules with the potential to induce this form of cell death in a therapeutic context. An
initial search for compounds reported to induce cellular vacuolization led us to an indolyl-
pyridinyl-propenone (IPP, also referred to as indole-derived chalcone) as a potential lead [13].

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Associated structure-activity relationship (SAR) studies revealed that the optimized scaffold for
induction of methuosis consists of a 2,5-disubstituted indole and a pyridine in the para-
Figure 1. Previously reported analogues illustrating the various biological activities of
substituted IPP’s.
configuration, bridged by an α,β-unsaturated ketone [11,14,15]. Our previously reported IPP
compounds, and their modes of biological activity, are summarized in Fig. 1.
To date, compounds 1a-1e are the most potent inducers of methuosis, possessing
activity between 2-3 ꢀM when assayed against the human glioblastoma cell line, U251. Among
these compounds 1a has been studied most thoroughly. Comparisons of structurally similar
IPP’s have revealed intriguing and unexpected results suggesting that the morphological
appearance of vacuoles in the treated cells is not always associated with cell death. For
instance, analogues with larger aliphatic substitutions (2e-2g) on the 2-indolyl position caused
vacuolization but had surprisingly less cytotoxicity than the vacuole-inducing compounds with
Me (1a) or Et (1b) at this position (Fig. 1) [11]. Similarly, certain 5-substituted analogues (2a-
2c), as well as the 2-des-methyl derivative 2d, also induced vacuole formation but were not

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cytotoxic [15]. While 5-methoxy (1a) and 5-propoxyindole (1e) analogues triggered cell death
by methuosis, their structurally similar counterparts, namely 5-ethoxy (2a) and 5-
isopropoxyindole (2b) caused cytoplasmic vacuolization without cell death. Studies are
currently underway with this series of analogues to explore the mechanistic basis for their
differential cytotoxicity.
Another novel insight into the biological effects of the IPP compounds was gathered
from derivatives containing electron-withdrawing functionalities at the 2-indolyl position [15].
Derivatives containing trifluoromethyl (3a) or alkyl carboxylate (3b-3d) substitutions caused
minimal to no vacuolization but remained highly cytotoxic. Morphologically, cells treated with
the latter series of compounds did not resemble cells undergoing methuosis. Instead, the cells
displayed features consistent with disruption of tubulin polymerization and microtubule
architecture. Cell cycle analysis demonstrated an accumulation of cells in the G2/M phase,
with eventual death by mitotic catastrophe. In this respect, 3a-3d were quite distinct from the
methuosis-inducing compounds, which did not disrupt microtubules or cause mitotic arrest at
the same concentrations. The redirection of cytotoxicity from methuosis to microtubule
disruption for derivatives 3a-3d was associated with a significant increase in growth inhibitory
potency.
While our previous synthetic work focused on substitution at either the 2- or 5-indole
positions, a lack of information exists for substitutions at the 4-, 6-, or 7- positions (Scheme 1).
We noted that the importance of a 5-methoxy group for either methuosis or microtubule
disruption is further dependent upon the electron withdrawing properties of the 2-substituent.
In the present study we have synthesized and evaluated methoxy isomers of 1a to
sequentially survey the 4-, 6- and 7-positions while initially holding the 2-position constant.
Upon finding significant anti-mitotic activity for the 6-position isomer 9b, we immediately
prepared its 2-trifluoromethyl version (15) by analogy to 3a where this type of functionality also
led to microtubule disruption [15]. Finally, drawing from several reports describing N-methyl-

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indole-based trimethoxyphenyl chalcones as compounds affecting tubulin polymerization, we
examined replacing the para-pyridine in our structural template with a trimethoxyphenyl group
and, likewise, separately examined the effect of adding a methyl group to the indole nitrogen.
The results reveal that the position of the methoxy group on the indole ring and the para-
pyridine are critical determinants of the biological activities of the IPP compounds.
2. Results and discussion
2.1. Chemistry
Scheme 1 illustrates the synthesis of isomers of 1a at the 4-, 6- or 7- indole position
(9a-9c). A disubstituted 5,6-dimethoxy derivative (9d) was also synthesized. From
commercially available 4a-4d, the indole nitrogen was protected with benzenesulfonyl chloride
(5a-5d). The benzenesulfonyl group ensured regioselective methylation at the 2-indolyl
position (6a-6d), which was accomplished under conditions of tert-butyllithium and
iodomethane [16]. Removal of benzenesulfonyl in a mixed solvent system of EtOH/aqueous
NaOH provided 7a-7d. Formylation reactions utilizing Vilsmeier conditions (8a-8d) followed by
Claissen-Schmidt condensation reactions produced target compounds 9a-9d. This approach
generally provided compounds in reasonable yields; however, intermediate 7b was not stable
under these conditions and resulted in low yields for 9b. When 9b was synthesized by the
alternative method shown in Scheme 2, intermediates were stable and produced high yields.
Compound 15 was also prepared according to Scheme 2. Targets 9b and 15 were
synthesized from aniline derivative 10, which was protected with BOC. Regioselective
acylation of 11 with Weinreb amide [17] 12a or 12b controlled by sec-butyllithium provided a
ketone intermediate, which subsequently cyclized to indole. The BOC protecting group was
cleaved with TFA to produce 7b or 13 [18]. Typical conditions of formylation (8b or 14) and
condensation afforded final targets 9b and 15.

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Scheme 1. Synthesis of either mono- or di-methoxyindole substituted IPP’s (9a-9d). Reagents
and conditions: (i) TBAB, 50% NaOH, THF; then benzenesulfonyl chloride, rt; (ii) THF, t-
o
o
o
butyllithium, -30 C to 0 C; then CH3-I, -30 C to rt; (iii) 3 M NaOH, EtOH, reflux; (iv) POCl3,
DMF, 0 oC to rt; then 1 N NaOH; (v) 4-Acetylpyridine, piperidine, MeOH, reflux.

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Scheme 2. Synthesis of trifluoromethyl compound 15 and a more efficient synthesis of 9b.
Reagents and conditions: (i) Boc2O, THF, reflux; (ii) THF, sec-butyllithium, -40 oC to 0 oC; then
Weinreb amide (12a or 12b)/THF, -40 oC to rt; (iii) DCM/TFA; (iv) POCl3, DMF, 0 oC to rt; then
1N NaOH; (v) 4-Acetylpyridine, MeOH, reflux.
The compounds synthesized in Scheme 3 probe the effects of a trimethoxyphenyl
functionality in place of para-pyridine. Aldehyde 16 was condensed with either 2,4,6-
trimethoxyacetophenone or 3,4,5-trimethoxyacetophenone to yield 17 or 18, respectively.
Piperidine is typically employed as the base to form the enolate from aryl acetates. This was
appropriate during the synthesis of 18, as well as target compounds 9a-9d and 15, but yielded
no reaction for 17. Presumably, the steric hindrance of methoxy groups present at the adjacent
2- and 6- positions of acetophenone prevented the formation of enolate by piperidine.
Compound 17 was synthesized using 50% aqueous KOH/MeOH, albeit in relatively low yields
as compared to other analogues in this series. Additionally, derivative 19 was synthesized

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from 1a in one step using sodium hydride as the base and methyl iodide as the alkylating
reagent (Scheme 4).
Scheme 3. Synthesis of indole-trimethoxyphenyl-propenones 17 and 18. Reagents and
conditions: (i) 2’,4’,6’-Trimethoxyacetophenone, KOH, MeOH/H2O, reflux; (ii) 3’,4’,5’-
Trimethoxyacetophenone, piperidine, MeOH, reflux.
Scheme 4. Synthesis of N-Methyl analogue 19. Reagents and conditions: (i) 1. DMF, NaH;
then CH3-I.
2.2. Biological activity
Previous studies with U251 glioblastoma cells have established that the sulforhodamine
B (SRB) colorimetric assay is useful for evaluating the loss of viable cells and for ranking the
relative potency [11,15]. This assay is sensitive to both methuosis and microtubule disruption.
Growth inhibitory activities for all compounds at a 48 h end-point are listed in Table 1 as the

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dose able to achieve 50% growth inhibition (GI50) compared to growth within control cultures
treated with only vehicle (DMSO).
Table 1. Summary of growth inhibition results and phenotypical analysis for final
targets.
Compound
GI50 (in ꢀM)*
2.32 ± 0.04
8.67 ± 1.23
0.09 ± 0.01
4.42 ± 0.39
>10
Vacuoles
1a
9a
9b
9c
9d
15
17
18
19
Abundant, Persistent
Few, Transient
Few, Transient
No
No
>10
No
3.74 ± 0.52
>10
No
No
>10
Few, Transient
*Results are the mean ± S.D. derived from three separate SRB assays. >10 indicates that
growth inhibition relative to control did not reach 50% at the highest concentration tested (10
ꢀM).
In the early stages of methuosis, cells become filled with a large number of phase-
lucent macropinosome-derived vacuoles, which can be readily observed by phase contrast
microscopy. As the cells begin to die (usually between 24-48 h), they detach from the culture
surface and lose membrane integrity. Phase-contrast microscopy pictures of cells treated with
the compounds at 2.5 ꢀM for 4 h or 48 h are compiled in Fig. 2. Consistent with our previous
studies, the 5-methoxy compound 1a induced methuosis with a GI50 of 2.30 ꢀM. By 48 h,
tumor cells treated with the compound at concentrations ≥ 2.5 ꢀM began to detach from the
dish and lose membrane integrity. By comparison, the 4-methoxy compound 9a elicited much
fewer vacuoles and possessed weak growth inhibitory activity. The 7-methoxy compound 9c
produced no vacuoles, but was moderately growth inhibitory. A strikingly different pattern of
activity was observed for the 6-methoxy compound 9b. Growth inhibitory activity increased
more than 25 fold, with a GI50 of 0.09 ꢀΜ. At the same concentration that 1a induced

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Figure 2. Morphological effects of the listed compounds on U251 glioblastoma cells. The control
cells received an equivalent volume of DMSO vehicle. Phase-contrast images were obtained at
the indicated time intervals after addition of compounds at 2.5 ꢀM.

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methuosis (2.5 µM), 9b caused early formation of
extensive membrane blebs on the cell surface and
general rounding of the cell body, with only a few
vacuoles detected (Fig. 2). By 24 h the majority of the
cells had rounded up and detached from the dish, and by
48 h most of the cells had disintegrated. The few
remaining attached cells were either rounded or enlarged
with multiple micronuclei. The higher potency and distinct
morphological characteristics observed with 9b were
similar to the effects we previously observed when
testing compounds 3a-3d in Fig. 1 [15]. Examination of
cells treated with 9b by immunofluorescence microscopy
with an antibody against tubulin (Fig. 3) confirmed
disruption of microtubules. Cells treated with 9b at 100
nM exhibited a dense network of microtubules, similar to
the DMSO control. However, at higher concentrations, 9b
caused a complete loss of normal microtubule
architecture in the few cells that remained attached to the
culture dish. In contrast, the microtubule network in cells
treated with the methuosis inducer, 1a, was generally
intact, except for distortions created by the presence of
the large cytoplasmic vacuoles.
Figure 3. Immunofluorescence
imaging of tubulin (red) in cells
treated for 24 h with 9b and 1a.
The nuclei are visualized with
DAPI (blue).
DNA histograms obtained by flow cytometry of
cells treated with 9b for 24 h (attached and detached
cells combined) were indicative of mitotic arrest at

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concentrations > 500 nM, with a large increase in the
G2/M phase population (Fig. 4). In contrast, cells
treated with 1a were predominantly in the G1/G0
phase of the cell cycle (Fig. 4). It is interesting to note
that cells treated with 100 nM 9b exhibited an intact
microtubule network (Fig. 3) and a near-normal cell
cycle distribution (Fig. 4), despite the growth inhibition
observed at this concentration in the SRB assay
(Table 1). This raises the possibility that at low
concentrations, near the GI50, 9b may inhibit cell
proliferation via an unidentified mechanism separate
from microtubule disruption. Based on the distinct
phenotypes elicited by 1a (methuosis) and 9b
(microtubule disruption), we were curious to determine
what type of activity might occur with a 5,6-
dimethoxyindole derivative, 9d. Surprisingly, the
dimethoxy compound lost all activity in the growth
assay and neither mode of action was observed in the
morphology assays (Table 1, Fig. 2).
We previously reported that the methuosis-
inducing activity of 1a could be switched to
Figure 4. DNA histograms of cells
microtubule-disrupting activity when trifluoromethyl
was substituted for methyl at the 2-position on the
indole ring (3a, Fig. 1) [15]. Therefore, we postulated
that a 2-trifluoromethyl substitution might increase the
treated with the indicated
compounds for 24 h were
generated by flow cytometry.

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activity of 9b. Interestingly, the resulting compound 15 lost all detectable growth inhibitory
activity (Table 1, Fig. 2).
The trimethoxyphenyl moiety and the indole scaffold are common motifs in many
compounds that possess anticancer activity mediated by microtubule disruption, including
several indole-based chalcones [19-23]. Features that distinguish the latter compounds from
our IPP series include methylation of the indolyl nitrogen and presence of either a 3,4,5-
trimethoxyphenyl [24,25] or 2,4,6-trimethoxyphenyl [26,27] group in place of the pyridine ring.
Based on these observations, we evaluated whether substitution of the pyridinyl moiety in 1a
with a trimethoxyphenyl moiety would switch activity from methuosis to microtubule disruption.
The results indicate that neither the 2,4,6-trimethoxyphenyl (17) nor the 3,4,5-trimethoxyphenyl
(18) derivative had any morphological effects on U251 cells that would be consistent with
methuosis or microtubule disruption (Fig. 2). Compound 18 had no detectable effect on cell
proliferation, whereas 17 was moderately growth inhibitory (Table 1). Similarly, we asked what
effect methylation of the indole nitrogen might have on the activity of potent methuosis inducer
1a. The resulting N-methyl derivative 19 (Scheme 4) showed substantially diminished growth
inhibition (GI50 >10 µM) and only transient vacuole-inducing activity at 2.5 µM (Fig. 2).
3. Conclusions
The present SAR studies demonstrate that the position of methoxy substitutions on the
indoly-pyridinyl-propenone scaffold have a significant influence on anti-cancer activity. The 5-
methoxy substitution is optimal for the induction of methuosis (1a). Changing the methoxy from
the 5-position (1a) to the 4-position (9a) or the 7-position (9c) of the indole ring attenuates or
eliminates methuosis activity. Unexpectedly, moving the methoxy group to the 6-position (9b)
provided a striking enhancement of growth inhibitory potency by conferring a different type of
anti-cancer activity to the compound; i.e., disruption of microtubules leading to mitotic arrest

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and cell death. Rather than enhancing either methuosis or microtubule-disrupting activity,
combining the 5- and 6-methoxy indole modifications so as to produce dimethoxy compound
9d essentially abolished both activities. Thus, two mutually distinct substitution patterns
emerge from our parent template, the 5-methoxy (1a) which induces methuosis and the 6-
methoxy (9b) which can disrupt microtubules without additionally having an electron
withdrawing substituent at the 2-position. Both types of arrangements are promising leads for
the development of new therapeutic agents that can remain effective when cancer cells
become resistant to apoptotic cell death induced by traditional anti-cancer drugs.
In addition to providing new insights regarding isomeric methoxy substitutions on the
indole, the present studies also reinforce the importance of the pyridinyl moiety for the
continued development of IPPs as potential anti-cancer therapeutics. Our previous studies
demonstrated that changing the configuration of the pyridinyl nitrogen (e.g., from para to meta)
in the context of various IPP’s with either methuosis or microtubule-disrupting activity
eliminated or markedly reduced activity [11,14,15]. Here we show that replacing the pyridine
ring with trimethoxyphenyl substituents (17 and 18) markedly reduced or eliminated the
morphological effects and growth inhibitory activity. These trimethoxyphenyl substitutions
previously were shown to impart potent microtubule-disrupting activity on various indole-based
chalcones containing N-methyl indole [24,25]. It thus appears that the trimethoxyphenyl
substituents are not compatible with the 5-methoxy-2-methylindole moiety for generating either
microtubule-targeted compounds or methuosis-inducing compounds.
Compounds that alter microtubule stability are widely used in cancer therapy [28-30].
However, these agents are not without drawbacks, such as dose-limiting toxicity, development
of drug resistance, and restricted penetrance of the blood-brain barrier [31,32]. Because of
these issues, there continues to be significant interest in discovery of new microtubule-
targeted compounds with distinct properties. In this regard, our findings suggest that further

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exploration of the anti-neoplastic potential and pharmacological properties of the 6-methoxy
derivative, 9b, could be productive.
4. Experimental section
4.1. Chemistry - General description
All reactions were performed in oven-dried 2-neck round-bottom flasks under an
atmosphere of either Ar or N2 and stirred with teflon-coated magnetic bars. TLC (silica gel F254
plates, Baker-flex) was used to monitor progress of all reactions with visualization performed
under 254 nm UV light. Reagent grade and anhydrous solvents were purchased from Sigma-
Aldrich and used without further purification unless otherwise noted. Compounds 4a-4d were
purchased from Alfa-Aesar, while compound 10 was purchased from Sigma-Aldrich.
Compounds 12a, 12b and 16 were reported previously [11,14,15]. Chromatography was
conducted on normal phase silica gel sorbent ( Fisher Scientific, 230-400 mesh) by flash
column methods as described previously [15,33] utilizing a gradient of increasing polar eluent
specifically indicated for each compound. Isocratic separations are denoted on an individual
basis. Samples to be purified were prepared by adsorption onto silica gel before performing
chromatography (previously described as “dry loading”) [15]. TLC was used to monitor product
elution during flash column chromatography. Appropriate fractions were combined, solvents
distilled in vacuo (rotary evaporator under water aspirator vacuum) and then further dried by a
vacuum pump (0.5 mm Hg) for 24 h unless described otherwise. Samples that were heated in
a vacuum desiccator were equipped to a vacuum pump (0.5 mm Hg) and dried for a specified
time and temperature denoted in the individual procedure. Solvent solutions dried with Na2SO4
were stored in a sealed flask and allowed to sit for at least 4 h. Upon completion, the drying
agent was removed by filtration and filtrate was evaporated in vacuo and then further dried by
a vacuum pump (0.5 mm Hg) for 24 h. Melting points were performed in triplicate on an

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Electrothermal digital melting point apparatus and are uncorrected. Proton (1H) and carbon
(13C) NMR experiments were recorded on either a 600 MHz Bruker Avance, Inova 600 MHz or
an Inova 400 MHz instrument. Samples were referenced to TMS when present, or the solvent
1
residual peak for H and 13C, respectively: (CDCl3; 7.27, 77.13; d6-DMSO; 2.50, 39.51; d6-
1
acetone; 2.05, 29.92). H NMR chemical shifts were given in ppm and coupling constants (J
values) were expressed in hertz (Hz) using the following designations: s (singlet), d (doublet), t
13
(triplet), dd (doublet of doublets), m (multiplet). The C chemical shifts were reported in ppm
for each compound in the experimental section and in all cases confirm structure. In a few
cases 13C shifts were found to double-up in their peak locations. Fluorine (19F) NMR was
recorded on an Inova 400 MHz instrument at 376 MHz. Samples were referenced externally to
CFCl3. Purity for tested compounds was determined by combustion analysis (Atlantic
Microlabs, Norcross GA) and HPLC. All tested compounds possess ≥95% as determined by
both purity methods. Observed values for combustion analysis were considered acceptable
within ±0.4% of calculated values. Synthetic derivatives reported as solvates are denoted in
the text. HPLC was performed on an Alliance® instrument (#2659) equipped with a quaternary
pump, an inline membrane degasser, autosampler and Photodiode Array (PDA) Detector
(#2996) from Waters Corporation (Milford, MA). The column was a Nova-Pak®C18 column, 4
µm particle size (150 mm × 3.9 mm). Samples were dissolved in 60% eluent A (H2O) and 40%
eluent B (CH3CN) for injection. The following procedure, termed “Gradient 1”, was employed
for final targets (9a-9d, 15, 17-19) and intermediate 8c: Time 0.01-2.00 min (isocratic, 20%
eluent B); Time 2.01-15.00 min (linear gradient, 45% eluent B to 80% eluent B); Time 15.01-
20.00 min (isocratic, 20% eluent B). Details for HPLC analysis are denoted in the individual
procedures. Chromatograms are illustrated in Figure S1 (supplementary data) and were
recorded at the UVmax for each compound.
4.2. Preparation of individual compounds

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4.2.1. 1-Benzenesulfonyl-4-methoxyindole (5a).
A suspension of 4-methoxyindole (500 mg, 3.4 mmol), TBAB (10 mol%, 109 mg, 0.34 mmol)
and 50% sodium hydroxide (5 mL) in THF (10 mL) and H2O (3 mL) was stirred vigorously for
20 min at rt. Benzenesulfonyl chloride (2 eq, 6.8 mmol, 1.2 g) in THF (15 mL) was added
dropwise to the reaction mixture. The reaction mixture was stirred overnight then extracted
with EtOAc (20 mL X 3). The combined organic layers were dried with anhydrous Na2SO4 and
then concentrated in vacuo to yield a yellow oil. The product was purified using column
chromatography (DCM) to yield white solid (910 mg, 92%): mp 86-88 oC. TLC Rf 0.71 (DCM).
1H NMR (600 MHz, CDCl3) δ 7.87-7.86 (d, 2H, J = 7.68 Hz), 7.61-7.59 (d, 1H, J = 8.34 Hz),
7.5155 (t, 1H, J = 7.38 Hz), 7.47 (d, 1H, J= 3.66 Hz), 7.43-7.41 (t, 2H, J = 7.8 Hz), 7.25-7.22 (t,
1H, J = 8.16 Hz), 6.78-6.77 (d, 1H, J = 3.66 Hz), 6.65-6.64 (d, 1H, J = 7.98 Hz), 3.88 (s, 3H).
13C NMR (150 MHz, CDCl3) δ 153.3, 138.4, 136.3, 134.0, 129.4, 127.0, 125.89, 124.97, 121.3,
106.68, 106.49, 103.7, 55.6. Elemental analysis calcd for C15H13NO3S: C, 62.70; H, 4.56; N,
4.87. Found: C, 62.71; H, 4.60; N, 4.97.
4.2.2. 1-Benzenesulfonyl-6-methoxyindole (5b).
A suspension of 6-methoxyindole (1.0 g, 6.79 mmol), TBAB (218 mg, 0.679 mmol) and 50%
sodium hydroxide (10 mL) in THF (10 mL) and H2O (3 mL) was stirred vigorously for 20 min at
rt. Benzenesulfonyl chloride (13.6 mmol, 2.4 g) in THF (8 mL) was added dropwise to the
reaction mixture. The reaction mixture was stirred overnight then extracted with EtOAc (50 mL
X 3). The combined organic layer was dried over Na2SO4 then concentrated in vacuo. The
product was purified using column chromatography (5% to 25% EtOAc/hexanes) to yield white
o
1
solid (1.80 g, 92%): mp 144-146 C (140-142 oC [34]). TLC Rf 0.38 (20% EtOAc/hexanes). H
NMR (600 MHz, CDCl3) δ 7.87-7.85 (m, 2H), 7.55-7.52 (m, 2H), 7.45-7.43 (m, 3H), 7.39-7.38
(d, 1H, J = 8.58 Hz), 6.87-6.85 (dd, 1H, J1 = 8.58 Hz, J2 = 2.34 Hz), 6.59-6.58 (dd, 1H, J1 =

ACCEPTED MANUSCRIPT
3.66 Hz, J2 = 0.72 Hz), 3.88 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 157.9, 138.2, 135.9, 133.8,
129.2, 126.7, 125.06, 124.45, 121.8, 112.6, 109.2, 97.9, 55.8.
4.2.3. 1-Benzenesulfonyl-7-methoxyindole (5c).
A suspension of 7-methoxyindole (1 g, 6.8 mmol), TBAB (219 mg, 0.68 mmol) and 50%
sodium hydroxide (10 mL) in THF (20 mL) and water (6 mL) was reacted and purified in a
o
manner similar to that for 5a to yield a white solid (1.5 g, 73%): mp 88-90 C (89-90 oC [35]).
TLC Rf 0.75 (DCM). 1H NMR (600 MHz, CDCl3) δ 7.85-7.84 (m, 3H), 7.57-7.54 (m, 1H), 7.49-
7.46 (m, 2H), 7.17-7.16 (m, 1H), 7.13–7.11 (m, 1H), 6.68-6.66 (d, 1H, J = 7.86 Hz), 6.66-6.65
(d, 1H, J = 3.66 Hz), 3.64 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 147.3, 140.4, 133.67, 133.11,
128.72, 128.59, 127.1, 124.55, 124.11, 114.0, 107.12, 106.76, 55.3.
4.2.4. 1-Benzenesulfonyl-5,6-dimethoxyindole (5d).
A suspension of 6-methoxyindole (1.01 g, 5.70 mmol), TBAB (184 mg, 0.570 mmol) and 50%
sodium hydroxide (5 mL) in THF (10 mL) and H2O (10 mL) was reacted and purified in a
o
similar manner to that for 5b to yield a white solid (1.71 g, 92%): mp 140-143 C (130-133 oC
1
[36]). TLC Rf 0.18 (20% EtOAc/hexanes). H NMR (600 MHz, CDCl3) δ 7.87-7.85 (m, 2H),
7.55-7.52 (m, 2H), 7.45-7.43 (m, 3H), 7.39-7.38 (d, 1H, J = 8.58 Hz), 6.87-6.85 (dd, 1H, J1 =
13
8.58 Hz, J2 = 2.34 Hz), 6.59-6.58 (dd, 1H, J1 = 3.66 Hz, J2 = 0.72 Hz), 3.88 (s, 3H). C NMR
(150 MHz, CDCl3) δ 157.9, 138.2, 135.9, 133.8, 129.2, 126.7, 125.06, 124.45, 121.8, 112.6,
1
109.2, 97.9, 55.8. H NMR (600 MHz, d6-acetone) δ 7.99-7.97 (m, 2H), 7.67-7.65 (m, 1H),
7.59-7.56 (m, 3H), 7.52 (d, 1H, J = 3.66 Hz), 7.08 (s, 1H), 6.67-6.66 (dd, 1H, J1 = 3.60 Hz, J2 =
0.66 Hz), 3.90 (s, 3H), 3.79 (s, 3H). 13C NMR (150 MHz, d6-acetone) δ 149.61, 148.71, 138.9,
135.1, 130.47, 130.06, 127.7, 126.0, 124.8, 110.7, 104.3, 98.4, 56.53, 56.30. Elemental
analysis calcd for C16H15NO4S: C, 60.55; H, 4.76; N, 4.41. Found: C, 60.40; H, 4.84; N, 4.52.
4.2.5. 1-Benzenesulfonyl-6-methoxy-2-methylindole (6b).

ACCEPTED MANUSCRIPT
Compound 5b (1.50 g, 5.22 mmol) was dissolved in THF (25 mL). The solution was cooled to -
o
30 C and treated with tert-butyllithium (1.7 M in pentane, 6.79 mmol, 4.0 mL) slowly to
maintain an internal temperature of -30 oC. After completion, the reaction mixture stirred for an
o
o
additional 30 min at -30 C, then allowed to warm to 0 C and stirred for another 20 min. The
o
reaction mixture was cooled to -30 C and iodomethane (15.7 mmol, 0.97 mL) was added
drop-wise. The reaction mixture was warmed to rt and stirred for 16 h. The reaction mixture
was concentrated in vacuo and redissolved in DCM (100 mL) then washed with saturated
NaHCO3 (75 mL X 3). The organic layer was dried over Na2SO4, the filtrate collected and
concentrated in vacuo to yield crude material which was purified by chromatography (0% to
o
20% EtOAc/hexanes) to yield a brown solid (984 mg, 63%): mp 68-71 C. TLC Rf 0.29 (10%
EtOAc/hexanes). 1H NMR (600 MHz, CDCl3) δ 7.76-7.74 (m, 3H), 7.54-7.51 (m, 1H), 7.43-7.41
(m, 2H), 7.27-7.26 (d, 1H, J = 8.58 Hz), 6.85-6.84 (dd, 1H, J1 = 8.52 Hz, J2 = 2.34 Hz), 6.26 (t,
1H, J = 0.84 Hz), 3.87 (s, 3H), 2.55 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 157.3, 139.2, 138.0,
136.0, 133.6, 129.3, 126.2, 123.4, 120.3, 112.3, 109.4, 99.4, 55.8, 15.8. Elemental analysis
calcd for C16H15NO3S: C, 63.77; H, 5.02; N, 4.65. Found: C, 63.88; H, 5.17; N, 4.57.
4.2.6. 1-Benzenesulfonyl-5,6-dimethoxy-2-methylindole (6d).
Compound 5d (1.70 g, 5.36 mmol) was reacted and purified in a manner similar to that for 6b
o
to yield a cream-colored solid (742 mg, 42%): mp 129-131 C. TLC Rf 0.23 (20%
EtOAc/hexanes). 1H NMR (600 MHz, d6-acetone) δ 7.88-7.86 (m, 2H), 7.73 (s, 1H), 7.68-7.65
(m, 1H), 7.58-7.55 (m, 2H), 6.96 (s, 1H), 6.38-6.37 (t, 1H, J = 0.84 Hz), 3.88 (s, 3H), 3.79 (s,
3H), 2.56 (s, 3H). 13C NMR (150 MHz, d6-acetone) δ 148.7, 139.8, 136.6, 135.0, 131.8, 130.5,
127.2, 123.9, 111.1, 103.4, 100.1, 56.60, 56.27, 15.9. Elemental analysis calcd for
C17H17NO4S: C, 61.62; H, 5.17; N, 4.23. Found: C, 61.34; H, 5.17; N, 4.19.
4.2.7. 4-Methoxy-2-methylindole (7a).

ACCEPTED MANUSCRIPT
Compound 5a (700 mg, 2.44 mmol) was dissolved in THF (25 mL) under an Ar (g)
atmosphere. The solution was cooled to -30 oC and then treated with tert-butyllithium (1.7 M in
o
pentane, 3.16 mmol, 1.86 mL) slowly to maintain an internal temperature of -30 C. After
completion, the reaction mixture was allowed to stir for an additional 30 min at -30 oC then was
allowed to warm up to 0 oC and stirred for another 20 min. The reaction mixture was cooled to
-30 oC and iodomethane (0.46 mL, 7.3 mmol) was added dropwise to the stirring mixture. After
the addition was complete, the reaction mixture was stirred and warmed to rt overnight. The
mixture was concentrated in vacuo and redissolved in DCM (75 mL) and washed with
saturated NaHCO3 (50 mL x 3). The organic layer was separated and then dried over
anhydrous sodium sulfate. The filtrate was collected and concentrated in vacuo to yield black
oil which was immediately taken to the next step. Crude 6a (1.1 g) was dissolved in a solution
o
of 3 N NaOH/EtOH (50 mL, 1:1) and heated at 90 C for 24 h. The reaction mixture was
concentrated in vacuo and extracted with DCM (50 mL X 3). The combined organic layers
were dried over Na2SO4. The filtrate was collected and concentrated in vacuo to yield a brown
oil. The product was purified using column chromatography (0% to 10% EtOAc/hexanes) to
o
1
yield a white solid (250 mg, 64%): mp 88 C. TLC Rf 0.34 (20% EtOAc/hexanes). H NMR
(600 MHz, CDCl3) δ 7.85 (s, 1H), 7.05-7.02 (t, 1H, J = 8.10 Hz), 6.94-6.93 (d, 1H, J = 7.68 Hz),
13
6.51-6.50 (d, 1H, J = 7.56 Hz), 6.31 (s, 1H), 3.94 (s, 3H), 2.44 (s, 3H). C NMR (150 MHz,
CDCl3) δ 152.5, 137.3, 133.4, 121.6, 119.3, 103.8, 99.7, 97.5, 55.3, 14.1. Elemental analysis
calcd for C10H11NO • 0.15 EtOH: C, 73.64; H, 7.14; N, 8.34. Found: C, 73.83; H, 7.23; N, 7.97.
4.2.8. 6-Methoxy-2-methylindole (7b, Scheme 2).
Compound 11 (1.20 g, 5.06 mmol) was dissolved in THF (15 mL) under an atmosphere of
o
Argon. The solution was cooled to -40 C over 10 min and sec-butyllithium (1.4 M, 7.93 mL)
o
was added slowly as to maintain an internal temperature of < -25 C. After reaching 1
equivalent of sec-butyllithium (~3.96 mL) the reaction mixture turned a bright yellow signifying

ACCEPTED MANUSCRIPT
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de-protonation of the amide nitrogen. The reaction mixture was then cooled to -50 C and a
solution of N-methoxy-N-methylacetamide 12a (575 mg, 5.57 mmol) in THF (3 mL) was added
o
over 5 min. The reaction mixture was warmed to -10 C over 30 min. The mixture was
partitioned between Et2O (75 mL) and 0.5 N HCl (75 mL). The aqueous layer was separated
and extracted an additional two times with Et2O (50 mL). The Et2O phases were combined and
washed with brine (75 mL) and then dried over Na2SO4 to yield a dark oil. The crude
intermediate was dissolved in DCM (20 mL). TFA (3 mL) was added to the mixture which was
then stirred at rt for 24 h. Upon completion, the reaction mixture was added to a separatory
funnel and washed with saturated NaHCO3 (50mL) followed by brine (50 mL). The organic
layer was separated, dried with Na2SO4 and concentrated in vacuo to provide a crude oil
which was purified by chromatography (0% to 20% EtOAc/hexanes) to yield a white solid (146
o
1
mg, 18%): mp 106-108 C. TLC Rf 0.31 (20% EtOAc/hexanes). H NMR (600 MHz, d6-
acetone) δ 9.77 (s, 1H), 7.27-7.26 (d, 1H, J = 8.46 Hz), 6.84 (d, 1H, J = 2.22 Hz), 6.62-6.61
13
(dd, 1H, J1 = 8.52 Hz, J2 = 2.28 Hz), 6.04 (m, 1H) 3.77 (s, 3H), 2.36 (s, 3H). C NMR (150
MHz, d6-acetone) δ 156.5, 138.2, 134.8, 124.4, 120.4, 109.4, 100.2, 95.1, 55.7, 13.6.
Elemental analysis calcd for C10H11NO: C, 74.51; H, 6.88; N, 8.69. Found: C, 74.67; H, 6.90;
N, 8.66.
4.2.9. 7-Methoxy-2-methylindole (7c).
Compound 5c (890 mg, 3.10 mmol) was reacted and purified in a manner similar to that for 7a
to yield a white solid (120 mg, 24%): mp 83 oC (83-83.5 oC [37]). TLC Rf 0.74 (DCM). 1H NMR
(600 MHz, CDCl3) δ 8.09 (s, 1H), 7.13-7.12 (d, 1H, J = 7.92 Hz), 6.99-6.96 (t, 1H, J = 7.86 Hz),
13
6.58-6.57 (d, 1H, J = 7.44 Hz), 6.18 (s, 1H) 3.93 (s, 3H), 2.41 (s, 3H). C NMR (150 MHz,
CDCl3) δ 145.4, 134.6, 130.3, 126.2, 119.9, 112.5, 101.11, 100.65, 55.3, 13.6.
4.2.10. 5,6-Dimethoxy-2-methylindole (7d).

ACCEPTED MANUSCRIPT
Compound 6d (0.732 g, 2.21 mmol) was dissolved in a solution of 3 N NaOH/EtOH (75 mL,
1:1) and refluxed at 90 oC for 60 h. The reaction mixture was then concentrated in vacuo and
extracted using DCM (50 mL X 3). The combined organic layers were dried over Na2SO4,
collected and then concentrated to provide a brown oil. The product was purified using column
chromatography (0% to 20% EtOAc/hexanes) to yield a yellow solid (352 mg, 83%): mp 94-95
oC (90-91 oC [38]). TLC Rf 0.15 (20% EtOAc/hexanes). 1H NMR (600 MHz, d6-acetone) δ 9.66
(s, 1H), 6.95 (s, 1H), 6.88 (s, 1H), 6.00-5.99 (m, 1H), 3.77 (s, 6H), 2.35 (s, 3H). 13C NMR (150
MHz, d6-acetone) δ 147.4, 146.1, 134.5, 132.0, 123.2, 103.8, 100.3, 96.3, 56.85, 56.63, 13.7.
4.2.11. 4-Methoxy-2-methylindole-3-carboxaldehyde (8a).
o
DMF (2 mL) was cooled to 0 C. POCl3 (0.5 mL) was added and the reaction mixture was
stirred for 10 min at 0 oC. A solution of 7a (220 mg, 1.37 mmol) in DMF (2 mL) was added to
the reaction mixture drop-wise over 10 min. The solution was stirred for an additional 40 min
while warming to rt, then slowly poured into ice-cold 1 N NaOH (50 mL) and stirred for 10 min.
o
The precipitate was collected, washed with ice-cold H2O and dried at 40 C for 24 h in a
o
vacuum desiccator yielding a tan solid (177 mg, 68%): mp 190-192 C. TLC Rf 0.71 (80%
1
EtOAc/hexanes). H NMR (600 MHz, d6-DMSO) δ 12.08 (s, 1H), 10.44 (s, 1H), 7.10-7.08 (t,
1H, J = 7.92 Hz), 7.02-7.01 (d, 1H, J = 7.98 Hz), 6.73-6.72 (d, 1H, J = 7.8 Hz), 3.92 (s, 3H),
2.65 (s, 3H). 13C NMR (150 MHz, d6-DMSO) δ 187.3, 153.2, 142.7, 136.0, 122.7, 116.4, 113.6,
104.8, 102.2, 55.1, 13.8. Elemental analysis calcd for C11H11NO2: C, 69.83; H, 5.86; N, 7.40.
Found: C, 69.59; H, 6.00; N, 7.27.
4.2.12. 6-Methoxy-2-methylindole-3-carboxaldehyde (8b).
Compound 6b (0.718 g, 2.38 mmol) was dissolved in a solution of 3 N NaOH/EtOH (75 mL,
1:1) and refluxed at 90 oC for 36 h. The reaction mixture was then concentrated in vacuo and
extracted using DCM (50 mL X 3). The combined organic layers were dried over Na2SO4,
collected and concentrated in vacuo to provide brown oil 7b (205 mg, 53%). TLC and NMR of

ACCEPTED MANUSCRIPT
7b suggested the sample was rapidly decomposing. The product was immediately taken to the
o
next step. DMF (2 mL) was cooled to 0 C. POCl3 (0.4 mL) was added and the reaction
mixture was stirred for 10 min. A solution of crude 7b (205 mg, 1.27 mmol) in DMF (1 mL) was
added to the reaction mixture dropwise over 10 min. The solution was stirred for an additional
2 h. The reaction mixture was added to ice-cold 1 N NaOH (40 mL) and stirred for 10 min. The
precipitate was collected, washed with ice-cold H2O and dried overnight in a vacuum
o
o
desiccator set at 40 C yielding a tan solid (50 mg, 21%): mp 219-222 C. TLC Rf 0.47 (75%
1
EtOAc/hexanes). H NMR (600 MHz, d6-DMSO) δ 11.79 (s, 1H), 9.99 (s, 1H), 7.89-7.88 (d,
1H, J = 8.58 Hz), 6.87 (d, 1H, J = 2.22 Hz), 6.79-6.78 (dd, 1H, J1 = 8.52 Hz, J2 = 2.28 Hz),
3.77 (s, 3H), 2.64 (s, 3H). 13C NMR (150 MHz, d6-DMSO) δ 184.0, 156.2, 147.8, 136.3, 120.7,
119.4, 113.7, 110.9, 95.1, 55.2, 11.4. Elemental analysis calcd for C11H11NO2: C, 69.83; H,
5.86; N, 7.40. Found: C, 69.74; H, 5.98; N, 7.23.
4.2.13. 6-Methoxy-2-methylindole-3-carboxaldehyde (8b, Scheme 2).
This compound was prepared from 7b-Scheme 2 (280 mg, 1.74 mmol) in a similar manner to
that for 8a except the reaction was stirred for 2 h while warming to rt to yield a tan solid (297
o
1
mg, 90%): mp 223-225 C. TLC Rf 0.57 (EtOAc). H NMR (600 MHz, d6-DMSO) δ 11.79 (s,
1H), 9.99 (s, 1H), 7.89-7.88 (d, 1H, J = 8.58 Hz), 6.87 (d, 1H, J = 2.22 Hz), 6.79-6.78 (dd, 1H,
J1 = 8.58 Hz, J2 = 2.28 Hz), 3.77 (s, 3H), 2.64 (s, 3H). 13C NMR (150 MHz, d6-DMSO) δ 184.0,
156.2, 147.8, 136.3, 120.7, 119.4, 113.7, 110.9, 95.1, 55.2, 11.4. Elemental analysis calcd for
C11H11NO2 • 0.15 H2O: C, 68.84; H, 5.93; N, 7.30. Found: C, 68.48; H, 5.90; N, 7.24.
4.2.14. 7-Methoxy-2-methylindole-3-carboxaldehyde (8c).
This compound was prepared from 7c (100 mg, 0.62 mmol) in a manner similar to that for 8a
o
1
to yield a light brown solid (97 mg, 83%): mp 209 C. TLC Rf 0.53 (80% EtOAc/hexanes). H
NMR (600 MHz, d6-DMSO) δ 12.10 (s, 1H), 10.03 (s, 1H), 7.62-7.61 (d, 1H, J = 7.80 Hz), 7.09-
13
7.06 (t, 1H, J = 7.86 Hz), 6.78-6.77 (d, 1H, J = 7.74 Hz), 3.93 (s, 3H), 2.65 (s, 3H). C NMR

ACCEPTED MANUSCRIPT
(150 MHz, d6-DMSO) δ 184.2, 147.8, 145.5, 126.9, 124.9, 122.6, 114.0, 112.5, 103.6, 55.1,
11.2. HPLC analysis: retention time = 3.338 min; peak area, 99.45%; eluent A, H2O; eluent B,
CH3CN; Gradient 1 over 20 min with a flow rate of 1 mL min-1 and detection at 215 nm;
injection of 10 ꢀL of 20 ꢀM 8c.
4.2.15. 5,6-Dimethoxy-2-methylindole-3-carboxaldehyde (8d).
o
DMF (2 mL) was cooled to 0 C. POCl3 (0.6 mL) was added and the reaction mixture was
stirred for 10 min at 0 oC. A solution of 7d (344 mg, 1.80 mmol) in DMF (2 mL) was added to
the reaction mixture drop-wise over 10 min. The solution was stirred for an additional 3 h at rt,
then slowly poured into ice-cold 1 N NaOH (50 mL) and stirred for 10 min. The solution was
transferred to a separatory funnel and extracted with EtOAc (50 mL X 4). The combined
organic layers were washed with brine (100 mL) and then dried over Na2SO4. The filtrate was
collected and concentrated in vacuo. The material was purified by chromatography (20% to
o
100% EtOAc/ hexanes) to yield a beige solid (368 mg, 93%): mp 204-208 C. TLC Rf 0.25
(75% EtOAc/hexanes). 1H NMR (600 MHz, d6-acetone) δ 10.64 (s, 1H), 10.10 (s, 1H), 7.69 (s,
13
1H), 6.98 (s, 1H), 3.83 (s, 3H), 3.80 (s, 3H), 2.69 (s, 3H). C NMR (150 MHz, d6-acetone) δ
184.4, 148.64, 148.00, 146.6, 130.8, 120.0, 115.6, 104.2, 96.3, 56.51, 56.44, 11.6. Elemental
analysis calcd for C12H13NO3: C, 65.74; H, 5.98; N, 6.39. Found: C, 65.47; H, 5.95; N, 6.28.
4.2.16. trans-3-(4-Methoxy-2-methyl-1H-indole-3-yl)-1-(4-pyridinyl)-2-propen-1-one (9a).
Compound 8a (100 mg, 0.53 mmol) was dissolved in anhydrous methanol (15 mL). 4-
Acetylpyridine (96 mg, 0.79 mmol) and piperidine (67 mg, 0.79 mmol) were added and the
solution was heated to reflux for 24 h. A precipitate slowly formed which was collected,
o
washed with ice-cold MeOH (50 mL) and dried at 40 C in a vacuum desiccator for 24 h to
o
yield a bright orange powder (143 mg, 92%): mp 234 C. TLC Rf 0.27 (80% EtOAc/hexanes).
1H NMR (600 MHz, d6-DMSO) δ 11.98, (s, 1H), 8.82-8.81 (m, 2H), 8.46-8.44 (d, 1H, J = 15.54
Hz), 7.84-7.83 (m, 2H), 7.44-7.41 (d, 1H, J = 15.54 Hz), 7.10-7.08 (t, 1H, J = 7.92 Hz), 7.00-

ACCEPTED MANUSCRIPT
13
6.99 (d, 1H, J = 7.98 Hz), 6.70-6.68 (d, 1H, J = 7.74 Hz), 3.93 (s, 3H), 2.64 (s, 3H). C NMR
(150 MHz, d6-DMSO) δ 188.1, 153.2, 150.5, 145.3, 141.68, 141.46, 136.9, 123.1, 121.1,
116.13, 115.46, 109.3, 104.8, 102.1, 55.2, 14.0. Elemental analysis calcd for C18H16N2O2 •
0.25 MeOH: C, 72.98; H, 5.70; N, 9.33. Found: C, 72.62; H, 5.51; N, 9.14. HPLC analysis:
retention time = 5.020 min; peak area, 98.99%; eluent A, H2O; eluent B, CH3CN; Gradient 1
over 20 min with a flow rate of 1 mL min-1 and detection at 224 nm; injection of 10 ꢀL of 20 ꢀM
9a.
4.2.17. trans-3-(6-Methoxy-2-methyl-1H-indole-3-yl)-1-(4-pyridinyl)-2-propen-1-one (9b).
This compound was prepared from 8b (50 mg, 0.26 mmol) in a manner similar to that for 9a to
yield a bright orange powder (33 mg, 43%): mp 250-252 oC. TLC Rf 0.39 (80%
EtOAc/hexanes). 1H NMR (600 MHz, d6-DMSO) δ 11.83, (s, 1H), 8.81-8.80 (m, 2H), 8.07-8.04
(d, 1H, J = 15.18 Hz), 7.96-7.94 (m, 3H), 7.43-7.40 (d, 1H, J = 15.18 Hz), 6.91-6.90 (d, 1H, J =
13
2.28 Hz), 6.84-6.82 (dd, 1H, J1 = 8.64 Hz, J2 = 2.34 Hz), 3.80 (s, 3H), 2.56 (s, 3H). C NMR
(150 MHz, d6-DMSO) δ 187.9, 156.0, 150.6, 144.96, 144.92, 139.4, 137.3, 121.43, 121.10,
119.6, 112.6, 110.44, 109.55, 95.3, 55.2, 11.8. Elemental analysis calcd for C18H16N2O2 • 0.7
H2O: C, 70.90; H, 5.75; N, 9.19. Found: C, 70.51; H, 5.32; N, 8.93. HPLC analysis: retention
time = 5.779 min; peak area, 98.44%; eluent A, H2O; eluent B, CH3CN; Gradient 1 over 20 min
with a flow rate of 1 mL min-1 and detection at 230 nm; injection of 10 ꢀL of 20 ꢀM 9b.
4.2.18. trans-3-(6-Methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (9b,Scheme
2).
This compound was prepared from 8b-Scheme 2 (252 mg, 1.33 mmol) in a manner similar to
o
that for 9a to yield an orange solid (338 mg, 87%): mp 248-250 C. TLC Rf 0.32 (80%
EtOAc/hexanes). 1H NMR (600 MHz, d6-DMSO) δ 11.83, (s, 1H), 8.81-8.80 (m, 2H), 8.07-8.04
(d, 1H, J = 15.18 Hz), 7.96-7.94 (m, 3H), 7.43-7.40 (d, 1H, J = 15.18 Hz), 6.91-6.90 (d, 1H, J =
13
2.28 Hz), 6.84-6.82 (dd, 1H, J1 = 8.64 Hz, J2 = 2.34 Hz), 3.80 (s, 3H), 2.56 (s, 3H). C NMR

ACCEPTED MANUSCRIPT
(150 MHz, d6-DMSO) δ 187.9, 156.0, 150.6, 144.96, 144.92, 139.4, 137.3, 121.43, 121.10,
119.6, 112.6, 110.44, 109.55, 95.3, 55.3, 11.8. Elemental analysis calcd for C18H16N2O2 •
0.025 H2O: C, 73.84; H, 5.53; N, 9.57. Found: C, 73.45; H, 5.52; N, 9.32. HPLC analysis:
retention time = 5.932 min; peak area, 97.97%; eluent A, H2O; eluent B, CH3CN; Gradient 1
over 20 min with a flow rate of 1 mL min-1 and detection at 229 nm; injection of 10 ꢀL of 20 ꢀM
9b.
4.2.19. trans-3-(7-Methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (9c).
This compound was prepared from 8c (76 mg, 0.40 mmol) in a manner similar to that for 9a to
o
yield a bright orange powder (104 mg, 89%): mp 265-266 C. TLC Rf 0.43 (80%
EtOAc/hexanes). 1H NMR (600 MHz, d6-DMSO) δ 12.10 (s, 1H), 8.81-8.80 (m, 2H), 8.09-8.06
(d, 1H, J = 15.24 Hz), 7.95-7.94 (m, 2H), 7.63-7.62 (d, 1H, J = 7.92 Hz), 7.44-7.41 (d, 1H, J =
15.18 Hz), 7.16-7.13 (t, 1H, J = 7.92 Hz), 6.82-6.81 (d, 1H, J = 7.74 Hz), 3.95 (s, 3H), 2.57 (s,
3H). 13C NMR (150 MHz, d6-DMSO) δ 188.0, 150.6, 145.80, 144.98, 144.91, 139.6, 127.2,
125.9, 122.26, 121.41, 113.09, 112.94, 109.9, 103.6, 55.3, 11.8. Elemental analysis calcd for
C18H16N2O2 • 0.875 H2O: C, 70.17; H, 5.81; N, 9.09. Found: C, 69.78; H, 5.88; N, 8.79. HPLC
analysis: retention time = 5.342 min; peak area, 98.91%; eluent A, H2O; eluent B, CH3CN;
Gradient 1 over 20 min with a flow rate of 1 mL min-1 and detection at 225 nm; injection of 10
ꢀL of 20 ꢀM 9c.
4.2.20. trans-3-(5,6-Dimethoxy-2-methylindol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (9d).
This compound was prepared from 8d (355 mg, 1.62 mmol) in a manner similar to that for 9a
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to yield a dark orange gum (438 mg, 83%): mp 222-223 C. TLC Rf 0.14 (80%
EtOAc/hexanes). 1H NMR (600 MHz, d6-DMSO) δ 11.75 (s, 1H), 8.81-8.80 (m, 2H), 8.07-8.05
(d, 1H, J = 15.24 Hz), 7.94-7.93 (m, 2H), 7.43 (s, 1H), 7.36-7.33 (d, 1H, J = 15.24 Hz), 6.94 (s,
13
1H), 3.88 (s, 3H), 3.80 (s, 3H), 2.55 (s, 3H). C NMR (150 MHz, d6-DMSO) δ 188.1, 150.6,
146.8, 145.76, 145.17, 143.8, 139.7, 130.5, 121.4, 118.4, 112.5, 109.6, 103.7, 95.6, 56.40,

ACCEPTED MANUSCRIPT
55.71, 12.0. Elemental analysis calcd for C19H18N2O3 • 0.075 H2O: C, 70.50; H, 5.65; N, 8.65.
Found: C, 70.07; H, 5.64; N, 8.39. HPLC analysis: retention time = 5.311 min; peak area,
96.50%; eluent A, H2O; eluent B, CH3CN; Gradient 1 over 20 min with a flow rate of 1 mL min-1
and detection at 222 nm; injection of 10 ꢀL of 20 ꢀM 9d.
4.2.21. N-Boc-5-methoxy-2-methylaniline (11).
Compound 10 (2.0 g, 14.6 mmol) and di-tert-butyl-dicarbonate (3.51 g, 16.1 mmol) in THF (50
mL) were heated to reflux for 20 h. The reaction mixture was concentrated in vacuo and re-
dissolved in DCM (75 mL). This mixture was washed with saturated NaHCO3 (75 mL), and
then brine (50 mL). The organic layer was separated, dried over Na2SO4 and concentrated in
vacuo to produce an oil which was purified by chromatography (0% to 20% EtOAc/hexanes) to
o
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yield a white solid (2.80 g, 81%): mp 79-80 C (76-80 C [39]). TLC Rf 0.55 (20%
1
EtOAc/hexanes). H NMR (600 MHz, CDCl3) δ 7.56 (s, 1H), 7.03-7.02 (d, 1H, J = 8.34 Hz),
6.56-6.54 (dd, 1H, J1 = 8.34 Hz, J2 = 2.64 Hz), 6.28 (s, 1H), 3.80 (s, 3H), 2.18 (s, 3H), 1.54 (s,
9H). 13C NMR (150 MHz, CDCl3) δ 158.8, 153.0, 137.4, 130.9, 118.3, 109.6, 105.6, 80.7, 55.6,
28.6, 17.1.
4.2.22. 6-Methoxy-2-trifluoromethylindole (13).
This compound was prepared from 11 (1.18 g, 4.97 mmol) in a manner similar to that for
compound 7b-Scheme 2 except Weinreb amide 12b (859 mg, 5.47 mmol) was employed to
obtain a white solid (446 mg, 42%): mp 91-95 oC. TLC Rf 0.32 (15% EtOAc/hexanes). 1H NMR
(600 MHz, CDCl3) δ 8.29 (s, 1H), 7.56-7.55 (d, 1H, J = 8.46 Hz), 6.89-6.87 (m, 3H), 3.86 (s,
13
2
3H). C NMR (150 MHz, CDCl3) δ 158.4, 137.3, 124.6 (q, JFC = 39 Hz), 123.0, 121.52 (q,
1JFC = 266 Hz), 120.97, 112.1, 104.6 (q, 3JFC = 3 Hz), 94.4, 55.8. 19F NMR (376 MHz, CDCl3) δ
-60.6 (s, 3F). Elemental analysis calcd for C10H8F3NO • 0.11 CH2Cl2: C, 54.09; H, 3.69; N,
6.24. Found: C, 54.54; H, 3.34; N, 5.78.

ACCEPTED MANUSCRIPT
4.2.23. 6-Methoxy-2-trifluoromethylindole-3-carboxaldehyde (14).
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DMF (2 mL) was cooled to 0 C. POCl3 (0.6 mL) was added and the reaction mixture was
stirred for 10 min at 0 oC. A solution of 13 (286 mg, 1.33 mmol) in DMF (2 mL) was added to
the reaction mixture drop-wise over 10 min. The solution was stirred for 30 min while warming
to rt and then heated to 80 oC for 3 h. The mixture was slowly poured into ice-cold 1 N NaOH
(50 mL) and stirred for 10 min. The solution was transferred to a separatory funnel and
extracted with EtOAc (50 mL X 4). The combined organic layers were washed with brine (100
mL) and then dried over Na2SO4. The filtrate was collected and concentrated in vacuo. The
material was purified by chromatography (10% to 50% EtOAc/hexanes) to yield a white solid
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(105 mg, 32%): mp 243-245 C. TLC Rf 0.19 (20% EtOAc/hexanes). 1H NMR (600 MHz, d6-
acetone) δ 11.91 (s, 1H), 10.29 (s, 1H), 8.21-8.20 (d, 1H, J = 8.82 Hz), 7.07 (d, 1H, J = 2.22
Hz), 7.03-7.01 (dd, 1H, J1 = 8.82 Hz, J2 = 2.28 Hz), 3.85 (s, 3H). 13C NMR (150 MHz, d6-
acetone) δ 184.7, 160.1, 137.7, 130.9 (q, 2JFC = 39 Hz), 124.3, 122.1 (q, 1JFC = 268 Hz), 119.6,
19
117.6, 115.5, 95.6, 55.9. F NMR (376 MHz, d6-acetone) δ -51.9 (s, 3F). Elemental analysis
calcd for C11H8F3NO2: C, 54.33; H, 3.32; N, 5.76. Found: C, 54.49; H, 3.32; N, 5.71.
4.2.24. trans-3-(6-Methoxy-2-trifluoromethyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (15).
Compound 14 (53 mg, 0.22 mmol) was dissolved in anhydrous MeOH (10 mL). 4-
Acetylpyridine (40 mg, 0.33 mmol) and piperdine (28 mg, 0.33 mmol) were added and the
mixture heated to reflux for 20 h. Upon completion, volatiles were evaporated in vacuo and the
sample was purified by column chromatography (30% to 70% EtOAc/hexanes). The resulting
solid was recrystallized from MeOH and dried at 40 oC in a vacuum desiccator for 36 h to yield
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1
a yellow solid (40 mg, 53%): mp 305-308 C. TLC Rf 0.37 (75% EtOAc/hexanes). H NMR
(600 MHz, d6-DMSO) δ 12.35, (s, 1H), 8.85-8.84 (m, 2H), 8.38 (s, 1H), 8.21-8.18 (d, 1H, J =
15.72 Hz), 7.98-7.97 (m, 2H), 7.87-7.85 (d, 1H, J = 15.72 Hz), 7.01-6.98 (m, 2H), 3.95 (s, 3H).
13C NMR (150 MHz, d6-DMSO) δ 189.0, 156.8, 150.8, 144.1, 141.7, 139.7, 125.0 (q, 2JFC = 38