Synthesis and evaluation of indole-based chalcones as inducers of methuosis, a novel type of nonapoptotic cell death

Article

pubs.acs.org/jmc

Synthesis and Evaluation of Indole-Based Chalcones as Inducers of

Methuosis, a Novel Type of Nonapoptotic Cell Death

Michael W. Robinson,^†,‡Jean H. Overmeyer,^†Ashley M. Young,^†Paul W. Erhardt,*^,‡

and William A. Maltese*^,†

^†Department of Biochemistry and Cancer Biology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, Ohio

43614, United States

^‡Center for Drug Design and Development, Department of Medicinal and Biological Chemistry, University of Toledo College of

Pharmacy, 2801 W. Bancroft Ave., Toledo, Ohio 43606, United States

S

* Supporting Information

ABSTRACT: Methuosis is a novel caspase-independent form of cell death in which

massive accumulation of vacuoles derived from macropinosomes ultimately causes cells

to detach from the substratum and rupture. We recently described a chalcone-like

compound, 3-(2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (i.e., MIPP),

which can induce methuosis in glioblastoma and other types of cancer cells. Herein,

we describe the synthesis and structure−activity relationships of a directed library of

related compounds, providing insights into the contributions of the two aryl ring systems

and highlighting a potent derivative, 3-(5-methoxy, 2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (i.e., MOMIPP) that

can induce methuosis at low micromolar concentrations. We have also generated biologically active azide derivatives that may be

useful for future studies aimed at identifying the protein targets of MOMIPP by photoaffinity labeling techniques. The potential

significance of these studies is underscored by the finding that MOMIPP effectively reduces the growth and viability of

Temozolomide-resistant glioblastoma and doxorubicin-resistant breast cancer cells. Thus, it may serve as a prototype for drugs

that could be used to trigger death by methuosis in cancers that are resistant to conventional forms of cell death (e.g., apoptosis).

progressively larger vacuoles. This eventually causes a decrease

in metabolic activity and rupture of the cell membrane.⁶Death

is considered to be nonapoptotic, as is it is not accompanied by

nuclear chromatin condensation, cell blebbing, or nucleosomal

DNA fragmentation. Methuosis is also caspase-independent, as

it cannot be prevented by broad-spectrum caspase inhibitors

such as zVAD-fmk.⁶

INTRODUCTION

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Despite recent advances in developing therapeutic agents that

target regulatory pathways unique to certain types of cancer

cells, the mainstays for postsurgical adjuvant therapy of many

tumors remain radiation and DNA alkylating agents. An

example is the highly malignant brain tumor, glioblastoma

multiforme (GBM), where the current standard of care involves

surgery when possible, followed by adjuvant therapy with

radiation and oral Temozolomide (TMZ).¹A limitation of the

latter approaches is that they work by damaging DNA,

triggering the intrinsic apoptotic pathway.²Because GBM

cells typically harbor mutations in tumor suppressor genes (e.g.,

PTEN, p53, and pRB), they are relatively insensitive to

apoptotic stimuli.^3,4Moreover, glioblastoma cells develop

resistance to alkylating agents by increasing their capacity to

repair DNA lesions.⁵We believe that it may be possible to

develop new approaches to treat such drug-resistant cancers

through the induction of alternative nonapoptotic forms of cell

death, which do not depend on DNA damage as a trigger.

Toward this end, we have defined a unique form of cell death

termed “methuosis”.^6,7

Methuosis was initially characterized in GBM cells, where

this form of cell death was triggered by ectopic expression of

activated Ras and Rac GTPases. However, the potential for

exploiting this nonconventional cell death pathway to kill

cancer cells that are refractory to apoptosis depends on the

identification of molecules with druglike properties that can

induce methuosis. We recently described a prototype chalcone-

related compound that can induce cell death with the hallmarks

of methuosis in both TMZ-resistant and nonresistant GBM

cells, as well as other cancer cell lines derived from breast,

colon, and pancreas.¹⁰Herein, we report synthesis and

structure−activity relationship (SAR) studies of a directed

library of related compounds leading to (1) the definition of

key features required for methuosis-inducing activity, (2) the

identification of a derivative with improved biological activity,

and (3) the development of an active azide analogue that may

be suitable for use as a photoaffinity probe in future target

identification efforts.

The hallmark of methuosis is the displacement of much of

the cellular cytoplasmic space by vacuoles derived from

macropinosomes.⁶The latter are formed when membrane

ruffles (lamellipodia) enclose pockets of extracellular fluid and

are internalized.^8,9In methuosis, impairment of the recycling

and lysosome-directed trafficking of macropinocytotic vesicles

locks them in an intermediate stage where they fuse to form

Received: July 27, 2011

Published: February 15, 2012

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Figure 1. Structures of compounds 1 and 2, initially found to be active inducers of methuosis¹⁰as compared with commercially available analogues

3−8, which failed to induce the hallmarks of methuosis in culture U251 GBM cells.

a

Scheme 1. Synthesis of Analogues of MIPP (Compound 2)

a

The specific substituents at R₁, R₂, R₃, and Ar for each numbered compound are listed in the chart. For A−D, the specific conditions and reagents

were as follows: (A) piperidine, CH₃OH, reflux, 19−89%. (B) BBr₃, CH₂Cl₂, −78 °C, 61 and 95% for 15 and 21, respectively. (C) POCl₃, DMF, 0

°C, 90%; piperidine, CH₃OH, reflux, 89%. (D) NaH, CH₃I, DMF, RT, 82%.

vacuolization of glioblastoma cells within a few hours and

substantial loss of cell viability within 48 h.¹⁰Chemical database

searches yielded additional compounds with >75% similarity to

1. Among these, compound 2 (Figure 1) was selected for

further study. It induced vacuoles that were larger and more

numerous than those produced by compound 1. Compound 2

was assigned the acronym MIPP, that is, 3-(2-methyl-1H-indol-

3-yl)-1-(4-pyridinyl)-2-propen-1-one. Detailed evaluation of the

biological effects of MIPP indicated that the form of cell death

induced by this compound matched the profile of methuosis.¹⁰

Furthermore, MIPP was effective in reducing the growth and

viability of GBM cells that were highly resistant to TMZ.¹⁰

Because concentrations of MIPP ≥ 10 μM were required to

effectively induce methuosis, we began to assemble a directed

library of MIPP-related compounds and conducted initial SAR

comparisons with the goal of identifying analogues with

improved potency. We began by comparing the methuosis-

inducing activity of MIPP with several closely related

compounds that were commercially available (compounds 3−

8 in Figure 1). The latter did not trigger cellular vacuolization

when added to U251 GBM cells at a concentration of 10 μM.

Examination of compounds 1−8 (Figure 1) suggested that the

RESULTS

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SARs. As we began to seek druglike small molecules that

could trigger methuosis, we noted a report from Kirchhausen

and colleagues¹¹in which they described a molecule termed

vacuolin-1, along with several other triazine-based compounds,

that were capable of inhibiting Ca²⁺-dependent lysosomal

exocytosis. Although these compounds induced marked cellular

vacuolization, they did not cause cell death. However, in the

Supporting Information included with this report, a structurally

distinct vacuole-inducing compound captured our attention

because of its resemblance to a class of molecules termed

chalcones. The structure of this compound is depicted in Figure

1 (compound 1). Chalcones consist of a 1,3-diphenyl-2-

propen-1-one framework that serves as a precursor for

flavonoid natural products. However, the term chalcones has

been applied more broadly to describe a number of synthetic

derivatives built on this framework.¹²Several chalcones have

been found to have significant anticancer activity but have not

been reported to induce vacuolization of cells.¹²⁻¹⁴This

prompted us to ask whether compound 1 might represent a

novel type of chalcone that can kill cancer cells by inducing

methuosis. We found that compound 1 caused extensive

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Table 1. Summary of SAR Studies Performed on MIPP (Compound 2) and Related Compounds Generated in Schemes 1 and 2

*

Results are expressed as percent of controls that received vehicle alone (DMSO). Values are the mean SD of quadruplicate (MTT) or triplicate

(colony formation) determinations.

specific relationship between an indole and a pyridinyl ring was

likely to be playing a significant role in determining activity.

Specifically, comparison of compound 1 (active) with

compound 7 (inactive) demonstrated the importance of the

pyridinyl ring, because replacement of the latter with a para-

methoxy phenyl ring rendered the compound inactive.

Therefore, an initial set of analogues was synthesized to

investigate the influence of the position of the pyridine nitrogen

on biological activity.

Analogues based on the α,β-unsaturated ketone core can be

prepared by Claisen−Schmidt condensations between indole-3-

carboxaldehydes and aryl ketones.¹⁵Condensation of aceto-

phenone or various acetyl-pyridines with indole-3-carboxalde-

hyde yielded compounds 9−12 (Scheme 1A). The activities of

these compounds were compared to MIPP at a concentration

of 10 μM using three criteria: (1) morphological vacuolization

of live cells assessed by phase contrast microscopy at 24 and 48

h; (2) cell viability at a 48 h end point assessed by 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)

assay, with fresh compound added after the first 24 h; and (3)

colony-forming assays (2 week end point) performed on cells

exposed to the compounds for 48 h. The results of this analysis

(Table 1) indicated that a para-nitrogen orientation of the

pyridine ring is a key feature required for activity. The meta and

ortho analogues 10 and 11, as well as the acetophenone

analogue 9, were all relatively ineffective at inducing methuosis

as compared to MIPP. In contrast, removal of the 2-methyl

from the indole ring of MIPP (compound 12) reduced but did

not eliminate activity.

We next explored the consequences of functionalizing the 5-

position of the indole ring using commercially available 5-

methoxy and 5-benzyloxy indole-3-carboxaldehydes to prepare

compounds 13 and 14, respectively. The 5-methoxy compound

13, in turn, was demethylated with BBr₃to afford the 5-OH

compound 15 (Scheme 1B). As shown in Table 1, the activities

of compounds 13 and 14 were similar to MIPP when compared

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Scheme 2. Analogues with 5′ Modifications of the Indole Ring Generated by Functionalizing the Indole Prior to Introduction of

a

the Pyridine Moiety

a

Conditions and reagents: Compound 22: BBr₃, CH₂Cl₂, −78 °C, 93%. Compound 23: methyl-4-(bromomethyl)benzoate, NaOH, TBAB, CH₂Cl₂,

RT, 63%. Compound 24: (1) POCl₃, DMF, 0 °C; (2) NaHCO₃, 90%. Compound 25: (1) POCl₃, DMF, 0 °C; (2) 5 N NaOH, 99%. Compound 26:

4-acetyl-pyridine, piperidine, MeOH, reflux, 34%. Compound 27: 4-acetyl-pyridine, piperidine, MeOH, reflux, 21%.

Figure 2. Comparison of the effects of MOMIPP (compound 19) and MIPP (compound 2) on growth and viability of U251 GBM cells. (A). One

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day after plating the cells in 96-well dishes, MOMIPP ( ) or MIPP ( ) was added at the indicated concentrations. Controls consisted of cells in

parallel wells treated with an equivalent volume of vehicle (DMSO). Medium containing fresh compound was added after 24 h, and MTT assays

were performed after a total 48 h of treatment. Each point represents the mean ( SD) from four separate wells, with the results expressed as percent

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of the mean of the parallel control wells. (B) U251 cells seeded in parallel 35 mm dishes were treated with MOMIPP ( ), MIPP ( ), or an

▲

equivalent volume of DMSO ( ), and cells were harvested for counting on each of three consecutive days. Each point is a mean SD from three

separate cultures.

by colony-forming assay and cell morphology, although they

were not as cytotoxic in the short-term growth/viability assay.

In contrast, the 5-OH substituent, compound 15, exhibited

greatly reduced biological activity in all of the assays. To

confirm that the location of the pyridine nitrogen was still

crucial for activity of the 5-methoxy-substituted compounds,

analogues 16 and 17 were generated. Loss of activity confirmed

the necessity of the para-nitrogen (Table 1).

1), consistent with the detrimental effect of the 5-OH

previously observed in compound 15.

One of the limitations for using MIPP under physiological

conditions is its sparing solubility in aqueous solutions.

Therefore, we explored the effects of modifying the 5-position

of the indole with a group that would add polarity at

physiological pH. Because our previous SAR studies showed

that there was some flexibility at the 5-position of the indole

ring (compare compounds 13 and 14), we designed an

analogue of 14 that added charge and also included a methyl at

the indole's 2-position. We envisaged that the OH analogue 21

could be alkylated with methyl 4-(bromomethyl)benzoate,

which could then be hydrolyzed to its acid to provide a highly

water-soluble analogue at pH 7.4. However, we were somewhat

surprised that no appreciable amount of product could be

produced by direct alkylation of 21. Multiple bases were

screened with varying pK_avalues, from K₂CO₃and Cs₂CO₃, as

well as triethylamine, tetramethylguanidine, 1,8-

diazabicyclo[5.4.0]undec-7-ene (DBU), and NaH. In all

reactions, alkylation at the pyridine nitrogen was observed.

Stronger bases such as TMG and NaH, even using 1 equiv,

produced appreciable indole-N-alkylation as well as pyridine

and indole-OH alkylation. The yield of singly alkylated product

at the 5-indole position was negligible. Thus, a new route was

designed by functionalizing the indole before introduction of

the pyridine moiety (Scheme 2). Commercially available 2-

methyl-5-methoxy-indole was demethylated with BBr₃to

provide 22. Alkylation with 4-methyl-(bromomethyl)-benzoate

under phase-transfer conditions afforded mono-O-alkylated

Because comparisons of compounds 12−15 versus MIPP

suggested that modifications at the 5- and 2-positions of the

indole ring both affect activity, we generated a 2-methyl-5-

methoxy analogue starting from commercially available 2-

methyl-5-methoxy-indole. Key intermediate 18 was synthesized

by a Vilsmeier−Haack formylation of 2-methyl-5-methoxyin-

dole, followed by coupling with 4-acetyl-pyridine to yield 19

(Scheme 1C). The inhibitory activity of this compound

exceeded that of MIPP in both the MTT viability assay and

the colony formation assay (Table 1). Alternatively, methyl-

ation of the indole-nitrogen of compound 13 with NaH/CH₃I

in dimethylformamide (DMF) (Scheme 1D) to yield 20

produced a compound that induced some cytoplasmic

vacuolization but had only modest effects on cell viability

(Table 1). Thus, after comparing compounds 13, 19, and 20, it

became clear that optimal activity is achieved when the 1- and

2-positions of the indole are occupied by H and methyl,

respectively. The 5-OH compound 21, made by BBr₃

demethylation of 19 (Scheme 1B), showed a marked reduction

in activity as compared to the 5-methoxy compound 19 (Table

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product 23.¹⁶After formylation with POCl₃/DMF, intermedi-

ates 24 and 25 were independently prepared. Workup in mild

base (NaHCO₃) produced the ester 24, while workup in 5 N

NaOH gave acid 25. Condensations with 4-acetyl-pyridine

provided the corresponding targets 26 and 27. However,

testing in glioblastoma cultures revealed that neither 26 nor 27

induced methuosis (Table 1).

Biological Activity of Compound 19 (MOMIPP) vs

Compound 2 (MIPP). The foregoing studies identified

compound 19 as the most potent for inducing methuosis.

Hereafter, we refer to this compound by the acronym

“MOMIPP” for 3-(5-methoxy, 2-methyl-1H-indol-3-yl)-1-(4-

pyridinyl)-2-propen-1-one. Superior activity of MOMIPP

versus compound 2 (MIPP) was confirmed in studies with

U251 glioblastoma cells, using MTT viability assays, cell growth

assays, morphological assessment, and colony-forming assays to

compare MOMIPP and MIPP. Figure 2A shows the dose−

response curves for the effects of the drugs on cell viability.

Each compound was added at the indicated concentration for 2

days, with medium and compounds replenished after the first

day. The relative IC₅₀for MOMIPP was 1.9 μM versus 4.8 μM

for MIPP. To obtain a measure of the comparative duration of

activity for each compound, their effects on cell growth and

survival were assessed by counting the number of cells in

parallel cultures treated for three consecutive days with 2.5, 5,

or 10 μM compound (Figure 2B). Unlike the viability studies,

in these experiments, the compounds were added at the

beginning of the study and were not replenished for the duration.

Under these conditions, MOMIPP was clearly more effective

than MIPP in reducing cell growth and viability. The reduction

of cell number in the cultures treated with MOMIPP coincided

with massive early vacuolization of the cells and loss of

nonviable cells from the substratum (Figure 3A,B). In contrast,

the cells treated with MIPP initially underwent vacuolization on

days 1 and 2 but tended to recover, especially at the 2.5 μM

concentration (Figure 3A). These studies demonstrate that a

single application of MOMIPP has a much more sustained

effect than MIPP on cell morphology and cell viability. The

difference between the MOMIPP and the MIPP was

underscored when colony-forming assays were used to evaluate

proliferative capacity and long-term cell viability (Figure 4).

MOMIPP was clearly more effective than MIPP in reducing

colony formation when cells were treated for 2 days (Figure

4A). If treatment was shortened to just 4 h, MOMIPP was still

more effective than MIPP, but higher concentrations of both

compounds were required to reduce colony formation (Figure

4B).

To determine if the methuosis-inducing compounds might

be effective for targeting TMZ-resistant glioblastoma, we used a

previously generated TMZ-resistant U251 cell line that was

essentially unaffected by concentrations of TMZ as high as 100

μM¹⁰(Figure 5A). As shown in Figure 5B, both MIPP and

MOMIPP were able to induce methuosis and reduce cell

viability in the TMZ-resistant cells, but the efficacy of

MOMIPP was clearly superior to MIPP. The ability of

MOMIPP to kill drug-resistant tumor cells by methuosis

extends beyond glioblastoma. For example, we observed similar

MOMIPP-induced vacuolization and loss of colony-forming

ability in both parental and doxorubicin-resistant MCF-7 breast

cancer cells (Figure 5C,D). To determine if the concentration

of MOMIPP that is maximally toxic in cancer cells might also

be cytotoxic to normal cells, we treated human mammary

epithelial cells (HMEC) with 10 μM MOMIPP. As shown in

Figure 3. Comparison of the abilities of MOMIPP and MIPP to

induce the morphological hallmarks of methuosis. One day after

plating, U251 GBM cells were treated with MOMIPP or MIPP at final

concentrations of 2.5 (A) or 10 μM (B). Controls received an

equivalent volume of vehicle (DMSO). Cells were observed by phase

contrast microscopy on three sequential days after addition of the

compounds, without changing the medium or replenishing the

compounds. Methuosis is characterized by extensive accumulation of

phase-lucent cytoplasmic vacuoles, with eventual cell rounding and

detachment from the substratum as viability is compromised.^6,10

Figure 5E, HMECs underwent extensive cytoplasmic vacuoliza-

tion within 24 h after addition of MOMIPP. By 48 h, cell

viability was reduced approximately 40% as compared with a

70% reduction seen in MCF7 breast carcinoma cells treated for

the same period at a similar cell density (Figure 5F). To further

assess the effects of MOMIPP on normal cells, we applied the

compound to human skin fibroblasts. As in all of the other cell

lines tested, 10 μM MOMIPP induced obvious vacuolization in

the fibroblasts by 24 h (Figure 5G). Similar to the HMECs,

normal fibroblasts plated at subconfluent density to maintain

exponential growth during exposure to MOMIPP exhibited a

40% decline in viability by 48 h. However, when the fibroblasts

were plated at a high initial density, so that they would be in

stationary phase at the time that MOMIPP was added, cell

viability was essentially unaffected (Figure 5H), even though

the cells underwent vacuolization (not shown). These

observations suggest that although MOMIPP can be cytotoxic

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with reactive groups that might be useful for photoaffinity-

based drug target discovery efforts.¹⁸

Initially, we designed a pseudobenzophenone analogue of

MIPP as a potential photoaffinity probe. Benzophenones are

commonly incorporated into active molecules for target

identification studies.¹⁸⁻²¹On the basis of the structure of

compound 14, we postulated that a 5-benzoyl analogue might

retain activity and function as a photoaffinity probe. The

benzophenone analogue (compound 30, benzoyl-MIPP) was

prepared in two steps, starting with acylation of the Vilsmeier−

Haack intermediate of 2-methylindole (Scheme 3A). With

unmethylated indole, this system has been shown to direct

acylation to the 5- and 6-positions.²²In our hands, acylation of

the Vilsmeier−Haack intermediate of 2-methylindole gave a 1:3

mixture of 5- and 6-benzoyl-2-methylindole-3-carboxaldehyde,

respectively (28 and 29). This regioselectivity was characterized

by 1D nuclear Overhauser effect (NOE) as described in the

Experimental Section. In the second step of the scheme, the

final compounds 30 and 31 were made by coupling the

aldehydes with 4-acetyl pyridine in the presence of piperidine.

Subsequent morphology and viability studies revealed that

unlike the benzyloxy derivative (compound 14), neither of the

benzophenone analogues (30 and 31) induced methuosis when

tested in U251 cells (data not shown).

Aromatic azides represent another photoreactive moiety

commonly used in photoaffinity labeling.^18,23−25Thus, we next

explored the possibility that an azide could be added to either

the 5- or the 6-position of the indole ring without loss of

methuosis-inducing activity, as the azide represents less of a

structure change from MOMIPP than the benzoyl group of

compound 30. The synthesis of compound 36 (5-azido MIPP)

was based on the directed nitration of 2-alkylindoles toward the

5-position,²⁶with further conversion to an azide by

diazotization (see Scheme 3B). 2-Methylindole was selectively

nitrated in concentrated sulfuric acid to yield 32 (see the

Experimental Section details for validation of regiospecificity),

which was then hydrogenated to amine 33. Under mild

diazotization conditions, the aminoindole was converted to the

aromatic azide 34.²⁷The azide was formylated to obtain 35 and

condensed with 4-acetyl-pyridine, providing 36, 5-azido-MIPP.

Separately, the same reaction scheme was performed on 2-

methyl-5-methoxyindole, wherein nitration led predominately

to the 6-nitrated product 37. The same sequence of reaction

steps described above was then completed to obtain 41, 6-

azido-MOMIPP.

Figure 4. Comparison of the abilities of MOMIPP and MIPP to

inhibit survival of U251 GBM cells in colony-forming assays. (A) Cells

were plated for colony-forming assays as described in the Experimental

Section. One day after plating the cells, MOMIPP ( ) or MIPP ( )

was added to the medium at the indicated concentrations, and cells

were maintained in the presence of the compounds for 48 h.

Thereafter, the compounds were removed, and colonies >50 cells were

counted after 2 weeks. Each point represents the mean ( SD) from

three separate dishes, with the results expressed as percent of the mean

of the parallel control dishes containing DMSO. (B) The effects

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MOMIPP ( ) and MIPP ( ) on colony formation were compared as

in panel A, except that cells were exposed to the compounds for only 4

h instead of 48 h.

to normal cells, the latter are somewhat less sensitive to the

compound than the cancer cell lines, U251 and MCF7. A

possible explanation for this is suggested by the differential

effects of MOMIPP on subconfluent versus confluent

fibroblasts, which imply that the disruptions of endosomal

trafficking typified by vacuolization may be relatively well

tolerated in cells that are not dividing as compared with cells

that are actively proliferating.

Synthesis and Biological Activity of Potential Photo-

affinity Labels. Our SAR studies showed that very subtle

changes to the structures of MIPP or MOMIPP (e.g.,

modifications of the pyridine ring) can abolish their capacity

to induce methuosis. Although chalcones are widely recognized

as electrophiles, the level of structural specificity required for

induction of methuosis suggests that the immediate effects of

MIPP and MOMIPP are most likely due to their interactions

with one or more distinct molecular targets. Several examples of

protein-specific modifications by electrophilic compounds have

been reported (see the Discussion). Therefore, an important

goal for the future is to identify the targets of methuosis-

inducing chalcones. A common approach for identifying drug

targets is affinity chromatography.¹⁷However, at this point, our

SAR studies have not revealed any nonessential sites that could

be linked to a bulky affinity tag (e.g., biotin) or tethered to a

solid matrix without loss of activity. Therefore, we focused on

generating minimally modified biologically active analogues

Evaluation of the biological activities of 36 (5-azido MIPP)

and 41 (6-azido MOMIPP) indicated that both compounds

retained methuosis-inducing activity as judged by both cellular

vacuolization (Figure 6A) and inhibition of colony formation

(Figure 6B,C) in U251 cells. Although not as potent as

MOMIPP, the 5-azido compound was clearly superior to the 6-

azido compound in terms of its biological activity.

DISCUSSION

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Methuosis is a newly discovered form of nonapoptotic cell

death that is triggered by alterations of macropinocytotic

vesicular trafficking, resulting in massive cellular vacuolization

and loss of cellular metabolic integrity.⁶We recently described

MIPP as a chalcone-like small molecule that is capable of

inducing methuosis in GBM and other cancer cell lines.¹⁰Here,

we present SAR studies that have led to the identification of a

5-methoxy analogue termed MOMIPP, which demonstrates

improved potency and stability in cell culture systems. We have

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Figure 5. MOMIPP effectively inhibits the viability of drug-resistant GBM and breast cancer cells. (A) TMZ-resistant U251 glioblastoma cells

(U251-TR) were derived as described previously.¹⁰The graph, reprinted from ref 10 with permission, shows that in contrast to the parental U251

cells, the viability of U251-TR cells is not reduced by treatment with TMZ. (B) The U251-TR cells were treated with the indicated concentrations of

MIPP or MOMIPP for 48 h and then subjected to colony-forming assays as described in the Experimental Section. Each point is based on colony

counts from three dishes (mean SD), with the results expressed as percent of the mean from parallel control dishes treated with an equivalent

volume of vehicle alone (DMSO). (C) Parental and doxorubicin-resistant (DoxR) MCF-7 breast cancer cells⁵⁶were treated with 10 μM MOMIPP

for 24 h and then examined by phase-contrast microscopy. (D) Long-term viability of parental or DoxR MCF-7 cells was assessed by colony-forming

assay after a 48 h of treatment with the indicated concentrations of MOMIPP. The results are the mean SD of determinations performed on three

parallel dishes. (E) Normal HMECs were treated with 10 μM MOMIPP or an equivalent volume of DMSO (control) and examined by phase-

contrast microscopy after 24 h. (F) MCF-7 cells or HMECs were plated in 96-well plates. After 48 h, while the cells were still subconfluent, fresh

medium containing 10 μM MOMIPP or an equal volume of vehicle (DMSO) was added. MTT viability assays were performed at a 48 h end point.

Values are the means ( SD) from four separate wells. (G) Normal human skin fibroblasts were treated with 10 μM MOMIPP or an equivalent

volume of DMSO (control) and examined by phase-contrast microscopy after 24 h. (H) Normal human fibroblasts were seeded in 96-well plates at

subconfluent density (1000 cells/well) or confluent density (10000 cells/well). After 24 h, fresh medium containing 10 μM MOMIPP or an equal

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Figure 5. continued

volume of vehicle (DMSO) was added. MTT viability assays were performed at a 48 h end point. Values are the means ( SD) from four separate

wells.

a

Scheme 3. Synthesis of 5′ and 6′ Azido Derivatives of MIPP

a

(A) Conditions and reagents: Compounds 28 and 29: (1) COCl₂, DMF, 1,2-DCE, 0 °C; (2) AlCl₃, benzoyl chloride, 0 °C RT, 13 and 41% for 28

and 29, respectively. Compound 30: 4-acetyl-pyridine, piperidine, MeOH, reflux, 13%. Compound 31: 4-acetyl-pyridine, piperidine, MeOH, reflux,

74%. (B) Conditions and reagents: Compound 32: NaNO₃, H₂SO₄, 0 °C, 95%. Compound 33: H₂, 10% Pd/C, EtOH, RT, 97%. Compound 34: (1)

NaNO₂; (2) NaN₃, 75% AcOH, 0 °C, 66%. Compound 35: POCl₃, DMF, 0 °C, 88%. Compound 36: 4-acetyl-pyridine, piperidine, MeOH, reflux,

39%. Compound 37: NaNO₃, H₂SO₄, 0 °C, 62%. Compound 38: H₂, 10% Pd/C, EtOH, RT, 99%. Compound 39: (1) NaNO₂; (2) NaN₃, 75%

AcOH, 0 °C, 53%. Compound 40: POCl₃, DMF, 0 °C, 69%. Compound 41: 4-acetyl-pyridine, piperidine, MeOH, reflux, 39%.

also generated active azido compounds that may be useful for

future studies aimed at identifying the protein targets of

MOMIPP.

we learned that the para-nitrogen orientation of the pyridine

ring is critical for activity. Analogues with a 2-pyridine

(compound 10), 3-pyridine (compounds 11 and 16), pyrazine

(compound 17), or phenyl (compound 9) were inactive.

In considering the potential mechanisms through which

MOMIPP induces cell death by methuosis, one possibility is

that it could be acting as an electrophile (i.e., Michael

acceptor). Compounds possessing electrophilic moieties that

render them potential substrates to cellular nucleophiles are not

often used in drug design because they can arbitrarily modify

many biomolecules. This can lead to off-target effects, including

the formation of immunoreactive haptens.²⁸The SAR studies

summarized in Table 1 show that while many of the

compounds in our series possess the α,β-unsaturated ketone

scaffold and could act as putative Michael acceptors, only

MOMIPP and a few other compounds were effective inducers

of methuosis at micromolar concentrations. Thus, it seems

unlikely that MOMIPP is inducing cell death via general

electrophilic modification of proteins. It remains possible that

MOMIPP may be functioning as a target-specific Michael

acceptor, with certain features of the molecule mediating

association with a specific protein, where proximity of the α,β-

unsaturated ketone to nucleophilic residues (e.g., cysteine) may

then promote covalent modification. Examples of such protein-

specific modifications have been reported for compounds that

bind to the receptor protein ErbB2,²⁹the TRPA1 ion

channel,³⁰the nuclear protein KSRP/FUBP2,³¹and sortase

enzymes in Gram-positive bacteria.³²These types of observa-

tions have spurred a general resurgence of interest in drugs that

work by covalent modification of their specific targets.³³

In the course of generating a directed library of compounds

for SAR studies, we carried out Claisen−Schmidt condensation

reactions between various indole-3-carboxaldehydes and

aromatic ketones catalyzed by piperidine, which efficiently

provided indole-chalcones. As piperidine acts as a catalyst, we

initially carried out the aldol condensations in the presence of

catalytic amounts of piperidine. However, we generally

observed higher yields when piperidine was used in excess. In

almost all instances, the products precipitated from solution

and were thus easily purified from excess catalyst (or starting

material) by simple rinsing.

Our SAR studies provided several useful insights into the

molecular features required for the methuosis-inducing activity

of this class of compounds. First, regarding the indole ring,

methylation of the nitrogen (compound 20) or removal of the

2-methyl group (compound 13) reduced but did not eliminate

activity. At the 5-position, different modifications had opposite

consequences. The activity of the 5-methoxy compound (19,

MOMIPP) was substantially improved, and the 5-benzyloxy

(compound 14) was similar to unsubstituted MIPP. In contrast,

modification of the 5-position with OH (compound 21), p-

methyl ester benzyloxyl (compound 26), and p-COOH

benzyloxyl (compound 27) all caused a major loss of activity.

Together, these findings demonstrate that there may be some

flexibility at these positions on the indole ring for future

attempts to further improve potency, create more water-soluble

derivatives, or tether the compound to an affinity matrix.

Regarding the second aryl system of the chalcone framework,

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Figure 6. continued

as percent of the mean from parallel control dishes treated with an

equivalent volume of vehicle alone (DMSO).

Compounds broadly classified as chalcones have been shown

to exhibit anticancer activity through multiple mechanisms,

including disruption of p53 interactions with MDM2 or

HDM2,^13,34inhibition of p-glycoprotein,³⁵⁻³⁷and disruption

of microtubule polymerization.³⁸⁻⁴⁰Many of the latter

compounds were designed as analogues of colchicine and

combretastatins, natural products known to bind β-tubulin. A

number of publications have since established that chalcones

and related molecules can act as antimitotic agents, and

substantial progress has been made in understanding their

SAR.^41,42While our active methuosis-inducing compounds

(e.g., MOMIPP) can be classified as chalcones, their specific

features are quite distinct from most of the antimitotic

chalcones previously described. The stringent structural

specificity for induction of methuosis, with dependence on

the specific substitution patterns of both the indolyl and the

pyridinyl moieties, appears to differentiate MOMIPP from

chalcones previously reported as antimitotic agents. We have

not observed mitotic arrest prior to loss of viability in cells

treated with these compounds (unpublished observation).

Conversely, massive endosomal vacuolization akin to what we

have observed with MOMIPP has not been reported with the

antimitotic chalcones. Of all of the cytotoxic chalcones

described in the literature, the one most related to MOMIPP

contains an unsubstituted indole ring linked to a 3,4,5-

trimethoxy phenyl moiety.¹⁴However, we found that

replacement of the 4-pyridine of MOMIPP with a 3,4,5-

trimethoxy phenyl group yielded a compound that did not

induce appreciable cellular vacuolization or death when applied

to U251 cells at 10 μM for 48 h (not shown). This is consistent

with the lack of tolerance for modifications to the 4-pyridinyl

moiety and suggests that the cytotoxicity previously reported

for the trimethoxyphenyl indolyl chalcone¹⁴probably was not

due to methuosis.

In general, cell death induced by antimitotic chalcones is

thought to occur by classical apoptosis, not methuosis. A

possible exception was noted in a recent report where a

chalcone-derivative termed “C2” may have induced death in

glioblastoma cells by a nonapoptotic mechanism involving

accumulation of autophagic vacuoles.⁴³However, as we have

previously reported, the vacuoles induced during methuosis

arise from macropinosomes and endosomes, which are distinct

from autophagosomes.^6,10It would be premature to rule out

the possibility that MOMIPP might bind tubulin in a manner

similar to colchicine and related chalcones, but so far, the

preponderance of evidence suggests that the compounds

described in this study act by a different mechanism to trigger

abnormal macropinocytosis, swelling of endosomal compart-

ments, and nonapoptotic cell death.¹⁰Ultimately, clarification

of this mechanism will depend on identification of the specific

molecular target(s) of MOMIPP and related compounds.

The future identification of the specific target(s) of

MOMIPP will be important for several reasons: (1) The

expression level or activity of the identified target(s) might have

predictive value for determining which types of tumors would

be most susceptible to the compound, (2) understanding the

function(s) of the proteins targeted by MOMIPP could be

helpful for assessing the potential toxicity to normal cells, and

Figure 6. Comparison of the effects of MOMIPP (compound 19), 5-

azido-MIPP (compound 36), and 6-azido-MOMIPP (compound 41)

on morphology and viability of U251 glioblastoma cells. (A) The

indicated compounds were added to the cells at concentrations of 2.5

or 10 μM, and the cells were observed by phase-contrast microscopy

after 48 h. (B and C) U251 cells were treated with varying

concentrations of the indicated compounds for 48 h, and long-term

cell viability was assessed by colony-forming assays. Each represents

the counts from three dishes (mean SD), with the results expressed

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visualized with UV light. Flash column chromatography was

performed using silica gel 230−400 μm mesh size. Melting

points (MP) are uncorrected. Nuclear magnetic resonance

(NMR) spectra were recorded on either a 600, 400, or 200

MHz instrument. Peak locations were referenced using the

residual solvent peak [7.26 and 77.16 for CDCl₃¹H and ¹³C,

respectively, and 2.50 and 39.51 for dimethylsulfoxide (DMSO)

¹H and ¹³C]. Proton coupling constants (J values) and signals

are expressed in hertz using the following designations: s

(singlet), d (doublet), br s (broad singlet), m (multiplet), t

(triplet), dd (doublet of doublets), and qd (quintet of

doublets). NMR spectra are available in the Supporting

Information. All synthesized compounds were at least 95%

pure as determined by elemental analysis (Atlantic Microlabs,

Norcross, GA).

(3) knowledge about the target protein(s) will facilitate analysis

of the drug binding site that could suggest modifications to

increase potency or specificity. In this respect, our finding that

incorporation of a photoreactive azide at the 5-position of the

indole ring of MIPP yields a derivative that retains good

methuosis-inducing activity (Figure 6) offers several avenues

for protein target identification using established strategies.

Aside from the photoreactive azide 36, MOMIPP's core

structure contains two other features that may render it

suitable for target identification studies, potentially bypassing

the need for incorporation of a photoreactive azide. First,

MOMIPP itself may be photoreactive and could behave

similarly to an excited benzophenone. The core scaffold of

MOMIPP is a pseudodiarylketone (as one side contains a

double vinylogous amide through the indole, while the other

contains a 4-pyridine) and could potentially exhibit photo-

excitation chemistry similar to that of a benzophenone. We are

currently pursuing experiments to characterize MOMIPP's

inherent photoreactivity and its ability to insert into other

molecules. The second characteristic of MOMIPP's core

structure that may render it suitable for target identification is

its electrophilic α,β-unsaturated ketone moiety, which may be

responsible for a covalent modification of a nucleophilic target

residue (e.g., cysteine). Thus, if MOMIPP is covalently binding

to its target protein(s), there may be no need for photoinitiated

reactive intermediate formation. This possibility is also

undergoing further study.

2-Methylindole-3-carboxaldehyde (2a).⁵¹To a dried 300 mL two-

neck round-bottom flask under argon at 0 °C, N,N-DMF (3 mL) was

added, followed by POCl₃(1.05 mL, 11.3 mmol). After this was stirred

for 10 min, 2-methylindole (1.23 g, 9.37 mmol) dissolved in DMF (6

mL) was added dropwise via an addition funnel under argon. After 2 h,

1 N NaOH (70 mL) was added slowly, upon which a white precipitate

formed. The solid was filtered and dried under vacuum, yielding 1.32 g

1

(89%) of white solid. H NMR (600 MHz, d₆-DMSO): δ 11.998 (s,

1H, N−H), 10.050 (s, 1H, CHO), 8.044−8.032 (d, J = 7.2, 1H,

indole-4H), 7.388−7.375 (d, J = 7.8, 1H, indole-7H), 7.183−7.135 (m,

2H, indole-5,6H), 2.679 (s, 3H, methyl). ¹³C NMR (150 MHz, d₆-

DMSO): δ 184.3, 148.6, 135.4, 125.6, 122.7, 121.9, 120.0, 113.7, 111.4,

11.5. Melting point: 195−200 °C (published:⁵¹197−200 °C). TLC

(ethyl acetate:hexanes 4:1) R_f= 0.39. Elemental analysis calculated for

C₁₀H₉NO: C, 75.45; H, 5.70; N, 8.80. Found: C, 75.23; H, 5.70; N,

8.93.

The key observation that MOMIPP effectively induced

methuosis in TMZ-resistant GBM cells, as well as doxorubicin-

resistant breast cancer cells, raises the possibility that further

development of this compound could lead to useful therapeutic

agents for treating cancers that are resistant to drugs that

commonly work by inducing apoptosis. Ultimately, deployment

of MOMIPP or related compounds as anticancer agents will

need to address some challenges. Preliminary studies indicate

that MOMIPP's ability to induce vacuolization is not restricted

to cancer cells (Figure 5E−H). However, the studies with

normal HMECs and skin fibroblasts suggest that the

consequences of vacuolization for cell viability are more severe

for rapidly dividing cancer cells than normal cells, particularly

when the normal cells enter stationary phase at high cell density

(Figure 5H). This raises a possibility that a therapeutic window

might be identified for selective effects on cancer cells. A

second challenge relates to the poor aqueous solubility of

MOMIPP and most of its active analogues. However, similar

solubility issues have been encountered with other hydrophobic

anticancer drugs (e.g., taxol, camptothecin) and have been

circumvented through the use of appropriate excipients⁴⁴or

novel nanoparticle^45,46or liposome^47,48based delivery strat-

egies. The latter strategies may offer the additional benefit of

allowing selective targeting of nonspecific agents by surface

modification of the delivery vehicle with tumor-specific

peptides or antibodies.^45,49,50Thus, given the observed

cytotoxic affects of MOMIPP on drug-resistant cancer cells

and the range of available options for its delivery in vivo, we

believe that MOMIPP can serve as a valuable prototype for

further preclinical testing.

trans-3-(2-Methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one

(2).⁵²To a dried 500 mL round-bottom flask under argon, 2-

methylindole-3-carboxaldehyde (400 mg, 2.51 mmol) was dissolved in

anhydrous MeOH (10 mL). 4-Acetyl-pyridine (305 μL, 2.76 mmol,

1.1 equiv) and piperidine (82 μL, 0.83 mmol) were added, and the

reaction was stirred under reflux. A red-orange precipitate gradually

formed, and after 12 h, this solid was isolated by filtration, rinsed with

chilled methanol, and dried under vacuum, producing 458 mg (69%)

1

of yellow solid. NMR: H NMR (600 MHz, d₆-DMSO): δ 12.017 (s,

1H, N−H), 8.816−8.806 (dd, J₁= 4.5, J₂= 2.1, 2H, pyr-2,6H), 8.121−

8.095 (d, J = 15.6, 1H, CCH), 8.077−8.063 (m, 1H, indole-4H),

7.975−7.965 (dd, J₁= 4.5, J₂= 1.5, 2H, pyr-3,5H), 7.489−7.464 (d, J =

15.6, 1H, CCH), 7.415−7.400 (m, 1H, indole-7H), 7.220−7.200

(m, 2H, indole-5,6H), 2.596 (s, 3H, methyl). ¹³C NMR (150 MHz, d₆-

DMSO): δ 188.0, 150.7, 145.6, 144.9, 139.5, 136.3, 125.8, 122.4,

121.50, 121.46, 120.4, 113.1, 111.7, 109.4, 11.9. Melting point: 268−

272 °C. TLC (ethyl acetate:hexanes 4:1) R_f= 0.18. Elemental analysis

calculated for C₁₇H₁₄N₂O: C, 77.84; H, 5.38; N, 10.68. Found: C,

77.96; H, 5.28; N, 10.59.

trans-3-(1H-Indol-3-yl)-1-phenyl-2-propen-1-one (9).¹⁵In a dried,

25 mL round-bottom flask under argon, indole-3-carboxaldehyde (300

mg, 2.07 mmol) was dissolved in anhydrous methanol (8 mL).

Acetophenone (240 μL, 2.07 mmol) and piperidine (100 μL, 1.00

mmol) were added. The reaction was stirred under reflux for 18 h. Ten

percent acetic acid was added (10 mL), precipitating 248 mg of a

crude yellow solid. This was recrystallized in 100% EtOH, filtered, and

dried under vacuum, yielding a pure, yellow solid (198 mg, 39%,

1

247.29 MW). H NMR (600 MHz, d₆-DMSO): δ 8.133−8.114 (m,

3H, phenyl-2,6H, indole-2H), 8.095−8.081 (m, 1H, indole-4H),

8.075−8.049 (d, J = 15.6, 1H, CCH), 7.668−7.642 (d, J = 15.6, 1H,

CCH), 7.655−7.629 (m, 1H, phenyl-4H), 7.581−7.556 (t, J = 1.5,

2H, phenyl-3,5H), 7.504−7.490 (m, 1H, indole-7H), 7.254−7.231 (m,

2H, indole-5,6H). ¹³C NMR (150 MHz, d₆-DMSO): δ 188.8, 139.1,

138.5, 137.5, 133.4, 132.4, 128.7, 128.2, 125.1, 122.8, 121.2, 120.4,

115.3, 112.8, 112.5. Melting point: 167−170 °C (published:¹⁵166−

167 °C). TLC (in 2:1 ethyl acetate:hexanes) R_f= 0.48. Elemental

analysis calculated for C₁₇H₁₃NO: C, 82.57; H, 5.30; N, 5.66. Found:

C, 82.19; H, 5.35; N, 5.63.

EXPERIMENTAL SECTION

■

Chemistry. General Methods. Reagents and starting

materials were obtained from commercial suppliers without

further purification. Thin-layer chromatography (TLC) was

done on 250 μm fluorescent silica gel 1B−F plates and

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trans-3-(1H-Indol-3-yl)-1-(2-pyridinyl)-2-propen-1-one (10).⁵²In-

dole-3-carboxaldehyde (200 mg, 1.38 mmol) was added to a dried 100

mL round-bottom flask under argon, and anhydrous methanol (8 mL)

was added. 2-Acetyl-pyridine (232 μL, 2.07 mmol) and piperidine (69

μL, 0.7 mmol) were added, and the reaction was stirred under reflux

for 24 h, after which still no precipitate had formed (AcOH did not

lead to precipitation). The crude reaction mixture was concentrated

and directly applied to a silica column for chromatography (ethyl

acetate:hexanes 1:1). The product was partially separated from the

aldehyde starting material (some product coeluted with aldehyde and

was discarded), and 80 mg (23%) of purified product was isolated.

stirred under reflux, during which a crude yellow solid precipitated.

After 15 h, 10% acetic acid (10 mL) was added to further the

precipitation. The solid was filtered and dried under vacuum, yielding a

pure, yellow solid (121 mg, 76%). ¹H NMR (600 MHz, d₆-DMSO): δ

8.821−8.811 (dd, J₁= 4.5, J₂= 1.5, 2H, pyr-H2,6), 8.163−8.159 (d, J =

2.4, 1H, indole-H2), 8.117−8.091 (d, J = 15.6, 1H, CCH), 7.953−

7.943 (dd, J₁= 4.5, J₂= 1.5, 2H, pyr-H3,5), 7.532−7.506 (d, J = 15.6,

1H, CCH), 7.489−7.485 (d, J = 2.4, 1H, indole-H4), 7.405−7.391

(d, J = 8.4, 1H, indole-H7), 6.902−6.883 (dd, J₁= 9.0, J₂= 2.4, 1H,

indole-H6), 3.866 (s, 3H, methyl). ¹³C NMR (150 MHz, d₆-DMSO):

δ 188.4, 155.2, 150.7, 144.9, 140.9, 134.2, 132.4, 126.0, 121.5, 114.3,

113.3, 112.7, 112.4, 102.4, 55.6. Melting point: 235−237 °C. TLC (in

4:1 ethyl acetate:hexanes) R_f= 0.20. Elemental analysis calculated for

C₁₇H₁₄N₂O₂: C, 73.37; H, 5.07; N, 10.07. Found: C, 73.55; H, 5.00;

N, 10.04.

1

NMR: H NMR (600 MHz, d₆-DMSO): δ 11.989 (s, 1H, N−H),

8.831−8.819 (m, pyr-H2), 8.220−8.194 (d, J = 15.6, 1H, CCH),

8.154−8.105 (m, 3H, CCH, indole-H2, pyr-H3), 8.052−8.027 (m,

1H, pyr-H4), 7.976−7.962 (m, 1H, indole-H4), 7.679−7.656 (m, 1H,

pyr-H5), 7.522−7.508 (m, 1H, indole-H7), 7.285−7.262 (m, 2H,

indole-H5,6). ¹³C NMR (150 MHz, d₆-DMSO): δ 188.2, 154.3, 149.1,

139.3, 137.7, 137.6, 134.2, 127.1, 125.1, 122.3, 122.2, 121.4, 120.1,

114.3, 113.1, 112.7. Melting point: 141−145 °C. TLC (ethyl

acetate:hexanes 4:1) R_f= 0.40. Elemental analysis calculated for

C₁₆H₁₂N₂O·0.1C₄H₈O₂: C, 76.62; H, 5.02; N, 10.90. Found: C, 76.41;

H, 5.03; N, 10.80.

trans-3-(5-Phenylmethoxy-1H-indol-3-yl)-1-(4-pyridinyl)-2-

propen-1-one (14). In a dried, 25 mL round-bottom flask under

argon, 5-benzyloxyindole-3-carboxaldehyde (100 mg, 0.40 mmol) was

dissolved in anhydrous methanol (3 mL). 4-Acetyl-pyridine (75 μL,

0.68 mmol) and piperidine (20 μL, 0.2 mmol) were added. The

reaction was stirred under reflux, during which a crude yellow solid

precipitated. The solid was filtered, rinsed with cold methanol, and

dried under vacuum. This crude product (107 mg) was purified from

residual aldehyde by column chromatography in ethyl acetate:hexanes

trans-3-(1H-Indol-3-yl)-1-(3-pyridinyl)-2-propen-1-one (11).⁵³In-

dole-3-carboxaldehyde (100 mg, 0.69 mmol) was added to a dried 100

mL round-bottom flask and dissolved in anhydrous methanol (5 mL).

3-Acetyl-pyridine (113 μL, 1.03 mmol, 1.5 equiv) and piperidine (69

μL, 0.7 mmol) were added, and the reaction was stirred under reflux.

After 12 h, the reaction was cooled to room temperature, upon which a

precipitate formed. The solid was filtered, yielding 54 mg. However,

there was significant aldehyde present on a crude NMR. This mixture

was dry-loaded onto silica and purified by column chromatography

(methylene chloride:methanol 9:1), producing 33 mg of pure, yellow

solid (19%). NMR: ¹H NMR (600 MHz, d₆-DMSO): δ 12.004 (s, 1H,

N−H), 9.292−9.289 (d, J = 1.8, 1H, pyr-H2), 8.808−8.797 (dd, J₁=

4.8, J₂= 1.8, 1H, pyr-H6), 8.467−8.447 (dt, J₁= 7.8, J₂= 2.1, 1H, pyr-

H4), 8.174 (s, 1H, indole-H2), 8.154−8.140 (dd, J₁= 6.6, J₂= 1.8, 1H,

indole-H4), 8.120−8.095 (d, J = 15.0, 1H, CCH), 7.665−7.639 (d, J

= 15.6, 1H, CCH), 7.607−7.585 (m, 1H, pyr-H5), 7.506−7.492

(dd, J₁= 6.9, J₂= 1.5, 1H, indole-H7), 7.268−7.223 (qd, J₁= 6.6, J₂=

1.5, 1H, indole-H5,6). ¹³C NMR (150 MHz, d₆-DMSO): δ 187.9,

152.7, 149.3, 139.9, 137.6, 135.7, 134.1, 133.7, 125.1, 123.9, 122.9,

121.3, 120.7, 115.0, 112.9, 112.5. Melting point: 192−194 °C

(published:⁵³191 °C). TLC (methylene chloride:methanol 9:1) R_f=

0.26. Elemental analysis calculated for C₁₆H₁₂N₂O: C, 77.40; H, 4.87;

N, 11.28. Found: C, 77.00; H, 4.80; N, 11.12.

1

(1:1 → 3:1 gradient), yielding pure, yellow solid (70 mg, 49%). H

NMR (600 MHz, d₆-DMSO): δ 8.837−8.827 (dd, J₁= 4.5, J₂= 1.5,

2H, pyr-2,6H), 8.148−8.143 (d, J = 3.0, 1H, indole-2H), 8.095−8.069

(d, J = 15.6, 1H, CCH), 7.932−7.922 (dd, J₁= 4.5, J₂= 1.5, 2H, pyr-

3,5H), 7.556−7.553 (d, J = 1.8, 1H, indole-4H), 7.523−7.511 (d, J =

7.2, 2H, phenyl-2,6H), 7.460−7.434 (d, J = 15.6, 1H, CCH),

7.411−7.370 (m, 3H, phenyl-3,5H, indole-7H), 7.330−7.306 (t, J =

7.2, 1H, phenyl-4H), 6.979−6.961 (dd, J₁= 8.4, J₂= 2.4, 1H, indole-

6H), 5.252 (s, 2H, methylene). ¹³C NMR (150 MHz, d₆-DMSO):

188.4, 154.1, 150.7, 144.9, 140.9, 137.7, 134.6, 132.5, 128.4, 127.7,

127.6, 125.8, 121.5, 114.2, 113.3, 113.1, 112.7, 104.1, 69.8. Melting

point: 218−221 °C. TLC (in 4:1 ethyl acetate:hexanes) R_f= 0.26.

Elemental analysis calculated (for C₂₃H₁₉N₂O₂·0.2C₆H₁₄·0.05H₂O): C,

78.02; H, 5.93; N, 7.52. Found: C, 77.69; H, 5.62; N, 7.24.

trans-3-(5-Hydroxy-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one

(15). To a dried two-neck 250 mL round-bottom flask under argon at

−40 °C, 13 (352 mg, 0.99 mmol) was partially dissolved in CH₂Cl₂

(30 mL). BBr₃(10 mL, 1.0 M in CH₂Cl₂, 10 mmol) was added

dropwise via an addition funnel under argon. After 4 h, the reaction

was poured onto ice and treated with 5 N NaOH until pH ≈ 12. The

aqueous solution was isolated and treated with 5 N HCl until pH ≈ 7,

forming a brown precipitate, which was extracted with ethyl acetate (3

× 40 mL). Extracts were combined, dried with Na₂SO₄, filtered,

concentrated, and dried under vacuum to yield 158 mg of an orange

solid (61%). NMR: ¹H NMR (600 MHz, d₆-DMSO): δ 11.840 (s, 1H,

N−H), 9.109 (s, 1H, O−H), 8.826−8.816 (dd, J₁= 4.5, J₂= 1.5, 2H,

pyr-2,6H), 8.063−8.058 (d, J = 3.0, 1H, indole-2H), 8.048−8.022 (d, J

= 15.6, 1H, CCH), 7.902−7.892 (dd, J₁= 4.5, J₂= 1.5, 2H, pyr-

3,5H), 7.349−7.346 (d, J = 1.8, 1H, indole-4H), 7.307−7.293 (d, J =

8.4, 1H, indole-H7), 6.762−6.744 (dd, J₁= 8.4, J₂= 2.4, 1H, indole-

H6). ¹³C NMR (150 MHz, d₆-DMSO): δ 188.2, 152.9, 150.6, 145.0,

141.4, 134.8, 131.7, 126.0, 121.3, 113.6, 113.1, 112.7, 112.3, 104.9.

Melting point: 262−268 °C. TLC (in ethyl acetate:hexane 4:1) R_f=

0.35. Elemental analysis calculated for C₁₆H₁₂N₂O₂·0.1H₂O: C, 72.22;

H, 4.62; N, 10.53. Found: C, 72.21; H, 4.42; N, 10.26.

trans-3-(1H-Indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (12).⁵³In

a dried, 25 mL round-bottom flask under argon, indole-3-

carboxaldehyde (300 mg, 2.07 mmol) was dissolved in anhydrous

methanol (8 mL). 4-Acetyl-pyridine (229 μL, 2.07 mmol) and

piperidine (100 μL, 1.00 mmol) were added. The reaction was stirred

under reflux; gradually, yellow product precipitated during the

reaction. After 14 h, the reaction was cooled to room temperature,

filtered, and washed with chilled methanol and hexanes. Drying under

1

vacuum for 2 h yielded a pure, yellow solid (343 mg, 67%). H NMR

(600 MHz, d₆-DMSO): δ 8.826−8.816 (dd, J₁= 4.2, J₂= 1.8, 2H, pyr-

H2,6), 8.186−8.182 (d, J = 2.4, 1H, indole-H2), 8.128−8.117 (m, 1H,

indole-H4), 8.121−8.095 (d, J = 15.6, 1H, CCH), 7.978−7.968 (dd,

J₁= 4.2, J₂= 1.8, 2H, pyr-H3,5), 7.592−7.566 (d, J = 15.6, 1H, C

CH), 7.512−7.498 (m, 1H, indole-H7), 7.267−7.243 (m, 2H, indole

H-5,6). ¹³C NMR (150 MHz, d₆-DMSO): δ 188.4, 150.7, 144.7, 140.9,

137.6, 134.6, 125.0, 123.0, 121.5, 121.4, 120.6, 114.6, 112.9, 112.6.

Melting point: 266−268 °C (published:⁵³257−258 °C). TLC (in 4:1

ethyl acetate:hexanes) R_f= 0.23. Elemental analysis calculated for

C₁₆H₁₂N₂O: C, 77.40; H, 4.87; N, 11.28. Found: C, 77.35; H, 4.84; N,

11.23.

trans-3-(5-Methoxy-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-

one (13). In a dried, 25 mL round-bottom flask under argon, 5-

methoxyindole-3-carboxaldehyde (100 mg, 0.57 mmol) was dissolved

in anhydrous methanol (3 mL). 4-Acetyl-pyridine (63 μL, 0.57 mmol)

and piperidine (30 μL, 0.3 mmol) were added. The reaction was

trans-3-(5-Methoxy-1H-indol-3-yl)-1-(3-pyridinyl)-2-propen-1-

one (16). In a dried, 50 mL round-bottom flask under argon, 5-

methoxyindole-3-carboxaldehyde (100 mg, 0.57 mmol) was dissolved

in anhydrous methanol (4 mL). 3-Acetyl-pyridine 63 μL, 0.57 mmol)

and piperidine (30 μL, 0.30 mmol) were added. The reaction was

stirred under reflux, during which a crude orange solid precipitated.

After 20 h, the solid was isolated by vacuum filtration, rinsed with

chilled methanol, and dried under vacuum, yielding a pure, orange

solid (98 mg, 60%). ¹H NMR (600 MHz, d₆-DMSO): δ 11.891 (s, 1H,

N−H), 9.276−9.273 (d, J = 1.8, 1H, pyr-2H), 8.798−8.790 (m, 1H,

pyr-6H), 8.440−8.421 (m, 1H, pyr-4H), 8.155−8.150 (d, J = 3.0, 1H,

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indole-2H), 8.113−8.088 (d, J = 15.0, 1H, CCH), 7.607−7.582 (d, J

= 15.0, 1H, CCH), 7.598−7.584 (m, 1H, pyr-5H), 7.500−7.497 (d,

J = 1.8, 1H, indole-4H), 7.397−7.383 (d, J = 8.4, 1H, indole-7H),

6.892−6.874 (dd, J₁= 8.4, J₂= 2.4, 1H, indole-6H), 3.867 (s, 3H,

methyl). ¹³C NMR (150 MHz, d₆-DMSO): δ 187.9, 155.1, 152.6,

149.3, 139.9, 135.7, 133.8, 133.7, 132.3, 126.1, 123.9, 114.7, 113.2,

112.7, 112.4, 102.6, 55.6. Melting point: 169−173 °C. TLC (in 4:1

ethyl acetate:hexanes) R_f= 0.17. Elemental analysis calculated

C₁₇H₁₄N₂O₂: C, 73.37; H, 5.07; N, 10.07. Found: C, 73.09; H, 5.10;

N, 10.01.

0.52 mmol, 60% in mineral oil, 1.2 equiv, unwashed). After this was

stirred for 5 min, the starting material (120 mg, 0.43 mmol) dissolved

in DMF (1 mL) was added slowly and stirred for 5 min until a

homogeneous red solution was formed. Methyl iodide (40 μL, 0.65

mmol, 1.5 equiv) was added slowly. After 2 h, saturated NH₄Cl (20

mL) and H₂O (20 mL) were added; this was extracted with ethyl

acetate (3 × 30 mL), dried with Na₂SO₄, filtered, and concentrated.

The crude product was purified by column chromatography

(methylene chloride:methanol 95:5), providing 103 mg (82%) of

1

solid. NMR: H NMR (400 MHz, d₆-DMSO): δ 8.821−8.806 (dd, J₁

= 4.4, J₂= 1.6, 2H, pyr-2,6H), 8.143 (s, 1H, indole-2H), 8.081−8.042

(d, J = 15.6, 1H, CCH), 7.950−7.935 (dd, J₁= 4.4, J₂= 1.6, 2H, pyr-

3,5H), 7.517−7.473 (m, 3H, CCH, indole-4,7H), 6.971−6.942 (dd,

J₁= 9.2, J₂= 2.4, 1H, indole-6H), 3.875 (s, 3H, o-methyl), 3.838 (s,

3H, n-methyl). ¹³C NMR (100 MHz, d₆-DMSO): δ 188.2, 155.5,

150.6, 144.9, 140.2, 137.3, 133.1, 126.5, 121.5, 114.2, 112.3, 111.9,

111.5, 102.9, 55.7, 33.4. Melting point: 178−182 °C. TLC (in

methylene chloride:methanol 95:5) R_f= 0.35. Elemental analysis

calculated for C₁₈H₁₆N₂O₂·0.1H₂O: C, 73.50; H, 5.55; N, 9.52. Found:

C, 73.34; H, 5.77; N, 9.25.

trans-3-(5-Methoxy-1H-indol-3-yl)-1-(pyrazine)-2-propen-1-one

(17). In a dried, 25 mL round-bottom flask under argon, 5-

methoxyindole-3-carboxaldehyde (75 mg, 0.43 mmol) was dissolved

in anhydrous methanol (4 mL). Acetyl-pyrazine (52 mg, 0.43 mmol)

and piperidine (23 μL, 0.23 mmol) were added. The reaction was

stirred under reflux, during which a crude, yellow solid precipitated.

After 3 h, the solid was isolated by vacuum filtration, rinsed with

chilled methanol, and dried under vacuum. This crude product was

recrystallized in EtOH (8 mL) to remove residual aldehyde, yielding a

1

pure, yellow solid (28 mg, 24%). H NMR (600 MHz, d₆-DMSO): δ

11.960 (s, 1H, N−H), 9.244 (s, 1H, pyr-2H), 8.905−8.873 (m, 2H,

pyr-4,5H), 8.204−8.178 (d, J = 15.6, 1H, CCH), 8.155 (s, 1H,

indole-2H), 7.987−7.961 (d, J = 15.6, 1H, CCH), 7.427−7.412 (m,

2H, indole-4,7H), 6.934−6.915 (dd, J₁= 9.0, J₂= 2.4, 1H, indole-6H),

3.853 (s, 3H, methyl). ¹³C NMR (150 MHz, d₆-DMSO): δ 187.3,

155.1, 148.8, 147.6, 144.0, 143.7, 140.2, 134.6, 132.5, 126.0, 113.4,

113.2, 112.8, 111.9, 102.9, 55.6. Melting point: 176−180 °C. TLC (in

4:1 ethyl acetate:hexanes) R_f= 0.30. Elemental analysis calculated

C₁₆H₁₃N₃O₂: C, 68.81; H, 4.69; N, 15.05. Found: 68.76; H, 4.64; N,

14.99.

trans-3-(5-Hydroxy-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one

(21). In a dried 250 mL two-neck round-bottom flask under argon, 19

(500 mg, 1.71 mmol) was partially dissolved in anhydrous CH₂Cl₂(35

mL) and placed at −78 °C. BBr₃(17 mL, 1.0 M in CH₂Cl₂, 17 mmol)

was added dropwise via an addition funnel under argon. After addition,

the reaction was warmed to room temperature, and stirring was

continued for 1 h. Ice water (50 mL) was added, followed by 1 N

NaOH until the pH ≈ 12 (∼60 mL). The CH₂Cl₂was further

extracted with 1 N NaOH (3 × 20 mL). The basic extracts were

neutralized with 8 N HCl to a neutral pH (∼8 mL), upon which the

product precipitated. The precipitate was filtered and dried under

5-Methoxy-2-methyl-1H-indole-3-carboxaldehyde (18).⁵⁴To a

dried two-neck 25 mL round-bottom flask at 0 °C, POCl₃(1.00 mL,

10.8 mmol) was added to N,N-DMF (2.5 mL). After 10 min of

stirring, 2-methyl-5-methoxyindole (1.45 mg, 9.00 mmol) dissolved in

DMF (5 mL) was added dropwise. After 45 min, 1 N NaOH (50 mL)

was slowly added, forming a white precipitate. The solid was isolated

by filtration, washed with cold H₂O, and dried under vacuum, yielding

1.52 g of white solid (90%). ¹H NMR (600 MHz, d₆-DMSO): δ

11.875 (s, 1H, N−H), 10.009 (s, 1H, CHO), 7.570−7.566 (d, J = 2.4,

1H, indole-4H), 7.282−7.268 (d, J = 8.4, 1H, indole-7H), 6.797−

6.778 (dd, J₁= 9.0, J₂= 2.4, 1H, indole-6H), 3.764 (s, 3H, o-methyl),

2.646 (s, 3H, c-methyl). ¹³C NMR (150 MHz, d₆-DMSO): δ 184.1,

155.5, 148.6, 130.0, 126.4, 113.7, 112.1, 111.8, 102.3, 55.2, 11.5.

Melting point: 188−192 °C (published:⁵⁴191−194 °C). TLC (ethyl

acetate:hexane 4:1) R_f= 0.37. Elemental analysis calculated for

C₁₁H₁₁NO₂: C, 69.83; H, 5.86; N, 7.40. Found: C, 69.65; H, 5.95; N,

7.25.

trans-3-(5-Methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-

propen-1-one (19). To a dried 250 mL two-neck round-bottom flask

under argon, 2-methyl-5-methoxy-1H-indole-3-carboxaldehyde (1.51

g, 7.98 mmol) was dissolved in anhydrous MeOH (30 mL). 4-Acetyl-

pyridine (1.32 mL, 11.97 mmol, 1.5 equiv) and piperidine (0.788 mL,

7.98 mmol) were added, and the reaction was stirred under reflux. An

orange solid gradually precipitated, and this was isolated by filtration,

rinsed with chilled MeOH, and dried under vacuum, yielding 2.08 g of

orange solid (89%). ¹H NMR (600 MHz, d₆-DMSO): δ 11.909 (s, 1H,

N−H), 8.812−8.802 (dd, J₁= 4.2, J₂= 1.8, 2H, pyr-2,6H), 8.097−

8.072 (d, J = 15.0, 1H, CCH), 7.947−7.937(dd, J₁= 4.2, J₂= 1.8,

2H, pyr-3,5H), 7.434−7.430 (d, J = 2.4, 1H, indole-4H), 7.374−7.349

(d, J = 15.0, 1H, CCH), 7.315−7.301 (d, J = 8.4, 1H, indole-7H),

6.851−6.833 (dd, J₁= 8.4, J₂= 2.4, 1H, indole-6H), 3.860 (s, 3H, o-

methyl), 2.572 (s, 3H, c-methyl). ¹³C NMR (150 MHz, d₆-DMSO): δ

188.1, 155.2, 150.6, 145.8, 145.1, 136.6, 131.0, 126.6, 121.5, 112.8,

112.3, 110.9, 109.3, 103.5, 55.6, 12.2. Melting point: 252−256 °C.

TLC (ethyl acetate:hexane 4:1) R_f= 0.16. Elemental analysis

calculated for C₁₈H₁₆N₂O₂: C, 73.95; H, 5.52; N, 9.58. Found: C,

73.76; H, 5.46; N, 9.47.

1

vacuum, providing 454 mg (95%) of a yellow solid. NMR: H NMR

(600 MHz, d₆-DMSO): δ 11.833 (s, 1H, N−H), 9.072 (s, 1H, O−H),

8.818 (s, 2H, pyr-2,6H), 8.064−8.039 (d, J = 15.0, 1H, CCH),

7.897 (s, 2H, pyr-3,5H), 7.342 (s, 1H, indole-4H), 7.282−7.257 (d, J =

15.0, 1H, CCH), 7.209−7.195 (d, J = 8.4, 1H, indole-7H), 6.687−

6.674 (d, J = 7.8, 1H, indole-6H), 2.542 (s, 3H, methyl). ¹³C NMR

(150 MHz, d₆-DMSO): δ 187.7, 153.0, 150.7, 145.9, 145.4, 140.0,

130.2, 126.8, 121.3, 112.2, 112.0, 111.6, 109.1, 105.2, 12.0. Melting

point: 296−299 °C. TLC (in ethyl acetate:hexane 4:1) R_f= 0.19.

Elemental analysis calculated for C₁₇H₁₄N₂O₂·0.1CH₂Cl₂: C, 71.55; H,

4.99; N, 9.79. Found: C, 71.89; H, 5.02; N, 9.67.

2-Methyl-1H-indol-5-ol (22).⁵⁵A dried 250 mL two-neck round-

bottom flask was charged with 2-methyl-5-methoxyindole (1.00 g, 6.20

mmol) and purged with argon, and CH₂Cl₂(30 mL) was added and

stirred vigorously until the indole was dissolved. After the reaction was

placed at −78 °C, BBr₃(37.2 mL, 1.0 M in CH₂Cl₂, 37.2 mmol, 6

equiv) was added dropwise via an addition funnel under argon. After

addition, the reaction was allowed to slowly warm to room

temperature. Thirty minutes after the cooling bath was removed, the

reaction was poured into ice water (∼50 mL) and saturated NaHCO₃

(50 mL), to a neutral pH. This was extracted with CH₂Cl₂(3 × 50

mL); the aqueous phase retained a yellow color and was acidified to

pH ∼3 (with 5 N HCl) and extracted with ethyl acetate (2 × 50 mL).

The combined organic extracts were washed with brine, dried with

Na₂SO₄, filtered, and concentrated to a brown oil. After it was dried

under vacuum for 6 h, 845 mg of a pure brown solid was isolated

1

(93%, 147.17 MW). H NMR (600 MHz, d₆-DMSO): δ 10.538 (s,

1H, N−H), 8.476 (s, 1H, O−H), 7.028−7.014 (d, J = 8.4, 1H, indole-

7H), 6.701−6.698 (d, J = 1.8, 1H, indole-4H), 6.487−6.468 (dd, J₁=

9.0, J₂= 2.4, 1H, indole-6H), 5.906 (s, 1H, indole-3H), 2.305 (s, 3H,

methyl). ¹³C NMR (150 MHz, d₆-DMSO): δ 150.4, 135.8, 130.6,

129.4, 110.6, 109.8, 103.3, 98.5, 13.5. Melting point: 131−134 °C

(published:⁵⁵134 °C). TLC (in 1:1 ethyl acetate:hexanes) R_f= 0.28.

Elemental analysis calculated for C₉H₈NO: C, 73.45; H, 6.16; N, 9.52.

Found: C, 73.09; H, 6.29; N, 9.28.

5-(4-Methylbenzoate)methoxy-2-methyl-1H-indole (23). In a 250

mL round-bottom flask, 2-methyl-1H-indole-5-ol (670 mg, 4.55

mmol) was partially dissolved in CH₂Cl₂(50 mL). Tetra-n-

butylammonium bromide (808 mg, 2.5 mmol) was added, followed

trans-3-(5-Methoxy-1-methyl-indol-3-yl)-1-(4-pyridinyl)-2-

propen-1-one (20). N,N-DMF (3 mL) was added to a dried 100 mL

two-neck round-bottom flask under argon containing NaH (21 mg,

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by NaOH (50 mL of a 5 N solution, 250 mmol) and methyl 4-

(bromomethyl)benzoate (1.15 g, 5.01 mmol, 1.1 equiv). After 8 h, the

organic layer was removed, and the aqueous phase was extracted with

CH₂Cl₂(1 × 30 mL). The combined extracts were washed with brine,

dried with Na₂SO₄, filtered, and concentrated to an oil. Purification by

column chromatography (1:3 ethyl acetate:hexane) provided 839 mg

of pure product (63%, 295.33 MW) [followed by the 3,5-doubly

alkylated product (115 mg, 5%)]. ¹H NMR (600 MHz, d₆-DMSO): δ

10.755 (s, 1H, N−H), 7.980−7.966 (d, J = 8.4, 2H, phenyl-3,5H),

7.602−7.588 (d, J = 8.4, 2H, phenyl-2,6H), 7.150−7.136 (d, J = 8.4,

1H, indole-7H), 6.982−6.978 (d, J = 2.4, 1H, indole-4H) 6.717−6.699

(dd, J₁= 8.4, J₂= 2.4, 1H, indole-6H), 6.005 (s, 1H, indole-3H), 5.157

(s, 2H, methylene), 3.849 (s, 3H, o-methyl), 2.331 (s, 3H, c-methyl).

¹³C NMR (150 MHz, d₆-DMSO): δ 166.1, 151.9, 143.6, 136.3, 131.4,

mg of pure product (34%). ¹H NMR (600 MHz, d₆-DMSO): δ 11.937

(s, 1H, N−H), 8.829−8.819 (d, J = 6.0, 2H, pyr-2,6H), 8.069−8.044

(d, J = 15.0, 1H, CCH), 7.971−7.957 (d, J = 8.4, 2H, phenyl-3,5H),

7.905−7.896 (d, J = 5.4, 2H, pyr-3,5H), 7.662−7.649 (d, J = 7.8, 2H,

phenyl-2,6H), 7.479−7.475 (d, J = 2.4, 1H, indole-4H), 7.326−7.312

(d, J = 8.4, 1H, indole-7H), 7.284−7.259 (d, J = 15.0, 1H, CCH),

6.938−6.920 (dd, J₁= 8.7, J₂= 2.1, 1H, indole-6H), 5.358 (s, 2H,

methylene), 3.838 (s, 3H, o-methyl), 2.561 (s, 3H, c-methyl). ¹³C

NMR (150 MHz, d₆-DMSO): δ 188.1, 166.0, 153.9, 150.6, 146.0,

145.1, 143.5, 139.5, 131.3, 129.3, 128.8, 127.4, 126.4, 121.4, 112.8,

112.4, 111.9, 109.3, 104.8, 69.2, 52.2, 12.1. Melting point: 236−239

°C. TLC (ethyl acetate:hexanes 4:1) R_f= 0.26. Elemental analysis

calculated for C₂₆H₂₂N₂O₄·0.25C₄H₈O₂: C, 72.31; H, 5.39; N, 6.25.

Found: C, 72.62; H, 5.64; N, 5.95.

trans-3-[5-((4-Carboxyphenyl)-methoxy)-1H-indol-3-yl)]-1-(4-pyr-

idinyl)-2-propen-1-one (27). In a dried 100 mL round-bottom flask

under argon, 25 (200 mg, 0.65 mmol) was partially dissolved in

anhydrous methanol (15 mL). 4-Acetyl-pyridine (107 μL, 0.97 mmol,

1.5 equiv) and piperidine (256 μL, 0.65 mmol) were added, and the

reaction was refluxed. After 24 h, the solid precipitate was isolated by

filtration, yielding 75 mg of product contaminated with aldehyde.

Because of both solubility and poor filtration, plenty of product

remained in the filtrate; thus, the filtrate was dry loaded onto silica and

purified by column chromatography (methylene chloride:methanol

129.3, 129.0, 128.7, 127.4, 111.0, 110.1, 102.8, 99.0, 69.0, 52.1, 13.4.

Melting point: 151−154 °C. TLC (in 1:1 ethyl acetate:hexanes) R_f=

0.49 (doubly alkylated product R_f= 0.42). Elemental analysis

calculated for C₁₉H₁₇NO₃: C, 73.20; H, 5.80; N, 4.74. Found: C,

73.02; H, 5.82; N, 4.55.

5-(4-Methylbenzoate)methoxy-2-methyl-1H-indole-3-carboxal-

dehyde (24). To a dried two-neck 250 mL round-bottom flask under

argon, POCl₃(325 μL, 3.5 mmol) was added to N,N-DMF (8 mL) at

0 °C. After this was stirred for 5 min, 23 (320 mg, 1.08 mmol)

dissolved in DMF (4 mL) was added dropwise. The yellow solution

was slowly warmed to room temperature, and after 1 h, saturated

NaHCO₃was added (50 mL), producing a white precipitate, followed

by 1 N NaOH (20 mL) to complete precipitation (direct workup with

NaOH resulted in roughly 1:1 mixture of ester product to ester-

hydrolyzed analogue). The solid was filtered, rinsed with cold H₂O,

1

9:1), yielding 85 mg of purified acid product (21%). H NMR (600

MHz, d₆-DMSO): δ 11.947 (s, 1H, N−H), 8.831−8.821 (dd, J₁= 4.2,

J₂= 1.8, 2H, pyr-2,6H), 8.073−8.047 (d, J = 15.6, 1H, CCH),

7.954−7.941 (d, J = 7.8, 2H, phenyl-3,5H), 7.904−7.894 (dd, J₁= 4.2,

J₂= 1.8, 2H, pyr-3,5H), 7.630−7.616 (d, J = 8.4, 2H, phenyl-2,6H),

7.479−7.476 (d, J = 1.8, 1H, indole-4H), 7.326−7.312 (d, J = 8.4, 1H,

indole-7H), 7.289−7.264 (d, J = 15.0, 1H, CCH), 6.939−6.920 (dd,

J₁= 9.0, J₂= 2.4, 1H, indole-6H), 5.349 (s, 2H, methylene), 2.562 (s,

3H, methyl). ¹³C NMR (150 MHz, d₆-DMSO): δ 188.1, 167.3, 154.0,

150.6, 146.0, 145.1, 142.8, 139.6, 131.2, 130.3, 129.5, 127.2, 126.4,

121.4, 112.8, 112.4, 111.9, 109.3, 104.8, 69.3, 12.1. Melting point:

269−273 °C. TLC (methylene chloride:methanol 9:1) R_f= 0.41.

Elemental analysis calculated for C_{2 5}H_{2 0}N₂O₄·0.25

CH₂Cl₂·0.25CH₃OH: C, 69.34; H, 4.91; N, 6.34. Found: C, 69.28;

H, 5.22; N, 6.72.

2-Methyl-5-benzoyl-indole-3-carboxaldehyde and 2-Methyl-6-

benzoyl-indole-3-carboxaldehyde (28 and 29). In a dried two-neck

250 mL round-bottom flask under argon, N,N-DMF (339 μL, 4.4

mmol) was added to 1,2-dichloroethane (8 mL). The reaction was

cooled to 0 °C, and oxalyl chloride (377 μL, 4.4 mmol) dissolved in

1,2-DCE (8 mL) was slowly added, forming a white heterogeneous

mixture. The mixture was allowed to warm to room temperature while

stirring. After 15 min, the reaction was cooled to 0 °C, and 2-

methylindole (577 mg, 4.0 mmol) dissolved in 1,2-DCE (8 mL) was

slowly added, forming a dark red solution. After 1 h, AlCl₃(1.96 g,

14.7 mmol) was added and stirred vigorously. Benzoyl chloride (510

μL, 4.4 mmol) dissolved in 1,2-DCE (4 mL) was slowly added, and

the reaction was warmed to room temperature and stirred overnight.

After 24 h, cold H₂O (50 mL) was added, followed by 5 N NaOH (10

mL), and the mixture was stirred. After 1 h, 5 N HCl (18 mL) was

added, and this was extracted with methylene chloride (3 × 50 mL).

The combined 1,2-DCE and methylene chloride extracts were

combined, dried with Na₂SO₄, filtered, and concentrated. The crude

product mixture was purified by column chromatography (ethyl

acetate:hexanes 1:1 → 4:1), yielding 5-benzoyl product 28 (142 mg,

13%) and 6-benzoyl product 29 (429 mg, 41%) (1:3 regioselectivity

for 5 vs 6 benzoylation).

1

and dried under vacuum, yielding 315 mg of white solid (90%). H

NMR (600 MHz, d₆-DMSO): δ 11.902 (s, 1H, N−H), 10.000 (s, 1H,

CHO), 7.991−7.977 (d, J = 8.4, 2H, phenyl-3,5H), 7.664−7.660 (d, J

= 2.4, 1H, indole-4H), 7.633−7.619 (d, J = 8.4, 2H, phenyl-2,6H),

7.304−7.290 (d, J = 8.4, 1H, indole-7H), 6.906−6.887 (dd, J₁= 9.0, J₂

= 2.4, 1H, indole-6H), 5.211 (s, 2H, methylene), 3.854 (s, 3H, o-

methyl), 2.646 (s, 3H, c-methyl). ¹³C NMR (150 MHz, d₆-DMSO): δ

184.0, 166.1, 154.3, 148.7, 143.2, 130.4, 129.3, 128.8, 127.4, 126.4,

113.6, 112.3, 112.2, 104.0, 69.0, 52.1, 11.5. Melting point: 224−227

°C. TLC: (in ethyl acetate:hexanes 4:1) R_f= 0.35. Elemental analysis

calculated for C₁₉H₁₇NO₄: C, 70.58; H, 5.30; N, 4.33. Found: C,

70.45; H, 5.41; N, 4.51.

5-(4-Benzoate)methoxy-2-methyl-1H-indole-3-carboxaldehyde

(25). To a dried two-neck 250 mL round-bottom flask under argon,

POCl₃(565 μL, 6.1 mmol) was added to N,N-DMF (12 mL) at 0 °C.

After this was stirred for 5 min, 23 (600 mg, 2.03 mmol) dissolved in

DMF (9 mL) was added dropwise. The yellow solution was slowly

warmed to room temperature. After 1 h, the reaction was cooled to 0

°C, and 5 N NaOH (90 mL) was added. After this was stirred for 30

min, 5 N HCl (95 mL) was added to precipitate the product, which

was filtered and dried overnight under vacuum, yielding 626 mg (99%)

of white solid. ¹H NMR (600 MHz, d₆-DMSO): δ 11.944 (s, 1H, N−

H), 10.001 (s, 1H, CHO), 7.961−7.947 (d, J = 8.4, 2H, phenyl-3,5H),

7.664−7.660 (d, J = 2.4, 1H, indole-4H), 7.590−7.576 (d, J = 8.4, 2H,

phenyl-2,6H), 7.304−7.290 (d, J = 8.4, 1H, indole-7H), 6.902−6.884

(dd, J₁= 8.4, J₂= 2.4, 1H, indole-6H), 5.194 (s, 2H, methylene), 2.647

(s, 3H, methyl). ¹³C NMR (150 MHz, d₆-DMSO): δ 184.0, 167.3,

154.4, 148.8, 142.5, 130.4, 129.4, 127.3, 127.2, 126.4, 113.6, 112.3,

112.2, 104.0, 69.1, 11.5. Melting point: 267−270 °C. TLC: (in ethyl

acetate:hexanes 4:1) R_f= 0.25.

trans-3-[5-((4-Methylbenzoate)methoxy)-1H-indol-3-yl)]-1-(4-

pyridinyl)-2-propen-1-one (26). In a dried 100 mL two-neck round-

bottom flask under argon, 24 (50 mg, 0.15 mmol) was partially

dissolved in anhydrous methanol (2 mL). 4-Acetyl-pyridine (26 μL,

0.23 mmol) and piperidine (7 μL, 0.075 mmol) were added, and the

reaction was refluxed. A yellow precipitate gradually formed, and after

24 h, the reaction was brought to room temperature, and the solid was

isolated by filtration, rinsed with cold methanol, and dried under

vacuum, yielding 36 mg. However, crude NMR showed 1:0.15

product:aldehyde. This mixture was dry loaded onto silica and purified

by column chromatography (ethyl acetate:hexanes 2:1), providing 22

2-Methyl-5-benzoyl-indole-3-carboxaldehyde (28). ¹H NMR

(600 MHz, d₆-DMSO): δ 12.371 (s, 1H, N−H), 10.073 (s, 1H),

CHO, 8.462 (s, 1H, indole-4H), 7.723−7.711 (m, 2H, phenyl-2,6H),

7.673−7.656 (m, 2H, indole-6H, phenyl-4H), 7.587−7.561 (t, J = 7.8,

2H, phenyl-3,5H), 7.553−7.539 (d, J = 8.4, 1H, indole-7H), 2.728 (s,

3H, methyl). ¹³C NMR (150 MHz, d₆-DMSO): δ 196.1, 184.8, 150.8,

138.3, 138.0, 132.1, 130.9, 129.5, 128.5, 124.9, 124.7, 123.4, 114.4,

111.6, 11.6. 1-D NOE: irradiation of peak at δ 12.371 (N−H): NOE

signal enhancement seen at δ 7.549 (C−H) and 2.728 (CH₃). The H

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peak at 7.549 has ortho coupling (J = 8.4), proving the benzoyl group

inserted at the 5-position, not 6. Separately, irradiation of peak at δ

7.540 (C−H with ortho coupling): NOE signal enhancement seen at δ

12.370 (N−H), again proving the proximity of N−H to an ortho-

coupled C₇−H; signal enhancement also seen at δ 7.66 for C₆−H (the

benzoyl 2H triplet peak at δ 7.574 was also irradiated by proximity,

thus signal enhancement was seen for the other benzoyl C−H peaks at

δ 7.72 and δ 7.67). Melting point: 227−230 °C. TLC (ethyl

acetate:hexanes 4:1) R_f= 0.32. Elemental analysis calculated for

C₁₇H₁₃NO₂·0.2C₄H₈O₂(trace ethyl acetate): C, 76.11; H, 5.24; N,

4.99. Found: C, 75.74; H, 4.94; N, 5.06.

135.4, 132.0, 130.6, 129.4, 129.2, 128.4, 123.1, 121.5, 119.9, 114.5,

114.1, 109.6, 12.3. Melting point: 267−270 °C. TLC (ethyl

acetate:hexanes 4:1) R_f= 0.29. Elemental analysis calculated for

C₂₄H₁₈N₂O₂·0.05H₂O: C, 78.48; H, 4.97; N, 7.63. Found: C, 78.08; H,

5.01; N, 7.50.

2-Methyl-5-nitro-1H-indole (32).²⁶In a 250 mL round-bottom

flask, 2.62 g of 2-methyl-indole (20 mmol) was dissolved in 20 mL of

H₂SO₄after vigorous stirring. In a separate flask, 1.87 g of NaNO₃

(1.1×, 22 mmol, 84.99 MW) was dissolved in 20 mL of H₂SO₄, also

after vigorous stirring, and added dropwise via addition funnel to the

indole. After addition, the reaction was stirred for another 10 min and

then poured into 400 mL of ice water, precipitating a yellow product.

The product was isolated via filtration and washed with cold water.

After 12 h of drying under vacuum, 3.35 g of yellow product was

isolated (95%). ¹H NMR (600 MHz, d₆-DMSO): δ 11.703 (s, 1H, N−

H), 8.4003−8.400 (d, J = 1.8, 1H, indole-4H), 7.916−7.898 (dd, J₁=

9.0, J₂= 1.8, 1H, indole-6H), 7.422−7.407 (d, 1H, J = 9.0, 1H, indole-

7H), 6.408 (s, 1H, indole-3H), 2.419 (s, 1H, methyl). ¹³C NMR (150

MHz, d₆-DMSO): δ 140.5, 140.0, 139.4, 128.0, 115.9, 115.7, 110.7,

101.6, 13.4. 1-D NOE: irradiation of peak at δ 7.422−7.407 (C₇−H):

NOE signal enhancement seen at δ 11.690 (N−H) and 7.916−7.897

(C₆−H). Irradiation of peak at δ 11.690 (N−H): NOE signal

enhancement seen at δ 7.422−7.407 (C₇−H) and 2.419 (C₂−CH₃)

(NOE observed between the C−H proton with ortho coupling and

N−H proton, thus proving NO₂inserted at indole C-5 vs C-6).

Melting point: 166−169 °C (published:²⁶176−176.5 °C). TLC (in

1:1 ethyl acetate:hexanes) R_f= 0.38. Elemental analysis calculated for

C₉H₈N₂O₂: C, 61.36; H, 4.58; N, 15.90. Found: C, 61.54; H, 4.63; N,

15.89.

2-Methyl-6-benzoyl-indole-3-carboxaldehyde (29). ¹H NMR

(600 MHz, d₆-DMSO): δ 12.323 (s, 1H, N−H), 10.116 (s, 1H,

CHO), 8.173−8.159 (d, J = 8.4, 1H, indole-4H), 7.776−7.774 (d, J =

1.2, 1H, indole-7H), 7.741−7.729 (m, 2H, phenyl-2,6H), 7.683−7.659

(t, J = 7.2, 1H, phenyl-4H), 7.627−7.611 (dd, J₁= 7.8, J₂= 1.8, 1H,

indole-5H), 7.583−7.558 (t, J = 7.5, 2H, phenyl-3,5H), 2.744 (s, 3H,

methyl). ¹³C NMR (150 MHz, d₆-DMSO): δ 196.3, 185.4, 152.2,

138.7, 135.3, 132.8, 131.8, 130.1, 129.8, 129.1, 124.4, 120.3, 114.7,

114.4, 12.5. 1-D NOE: irradiation of peak at δ 12.323 (N−H): NOE

signal enhancement seen at δ 7.775 (C−H with meta coupling, thus

proving benzoyl addition to position 6 of indole) and δ 2.744 (CH₃).

Separately, irradiation of the peak at δ 7.775 (C−H with meta

coupling: J = 1.2) led to signal enhancement at δ 12.323 (N−H peak),

thus proving proximity of meta-coupled C−H to N−H (the benzoyl

2,6 2H peak at 7.73 was also irradiated by proximity, leading to signal

enhancement of benzoyl 3,5 2H peak at δ 7.57). Melting point: 192−

196 °C. TLC (ethyl acetate:hexanes 4:1) R_f= 0.40. Elemental analysis

calculated for C₁₇H₁₃NO₂: C, 77.55; H, 4.98; N, 5.32. Found: C,

77.28; H, 4.97; N, 5.15.

2-Methyl-5-amino-1H-indole (33).²⁶Compound 32 (1.00 g, 5.68

mmol) was dissolved in 75 mL of ethanol. 10% Pd/C was added (200

mg), and the mixture was subjected to H₂(38 psi) using a Parr

hydrogenator for 3.5 h. The mixture was filtered over Celite, which

was washed with methanol. After concentration and drying under

trans-3-(5-Benzoyl-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-

propen-1-one (30). In a dried 100 mL round-bottom flask under

argon, 28 (50 mg, 0.19 mmol) was dissolved in anhydrous methanol

(3 mL). 4-Acetyl-pyridine (21 μL, 0.19 mmol) and piperidine (4 μL,

0.04 mmol) were added, and the reaction was refluxed. After 12 h, 0.3

equiv of 4-acetyl-pyridine (6.3 μL, 0.06 mmol) was added. After a total

of 24 h of reaction time, the reaction was cooled, and the precipitate

was isolated by filtration and rinsed with cold methanol, providing 37

1

vacuum, 801 mg of a brown powder (97%) was isolated. H NMR

(600 MHz, d₆-DMSO): δ 10.370 (s, 1H, N−H), 6.943−6.929 (d, J =

8.4, 1H, indole-7H), 6.552−6.549 (d, J = 1.8, 1H, indole-4H), 6.373−

6.355 (dd, J₁= 8.4, J₂= 2.4, 1H), 5.815 (s, 1H, indole-6H), 4.436 (bs,

2H, NH₂), 2.288 (s, 3H, methyl). ¹³C NMR (150 MHz, d₆-DMSO): δ

140.8, 135.0, 129.9, 129.6, 110.5, 110.3, 102.9, 98.0, 13.5. Melting

point: 147−150 °C (published:²⁶157−159 °C). TLC (in 3:1 ethyl

acetate:hexanes) R_f= 0.30; (in 1:1 ethyl acetate:hexanes) R_f= 0.16.

Elemental analysis calculated for C₉H₁₀N₂·0.05C₂H₆O: C, 73.61; H,

6.99; N, 18.87. Found: C, 73.48; H, 6.77; N, 18.81.

2-Methyl-5-azido-1H-indole (34). In an oven-dried, 250 mL round-

bottom flask flushed with argon, 33 (400 mg, 2.74 mmol, 146.19 MW)

was dissolved in 90% AcOH (20 mL). After complete solvation, the

reaction was placed at 0 °C and protected from light with foil. NaNO₂

(1.1×, 3.01 mmol, 208 mg, 69.00 MW) dissolved in cold H₂O (2 mL)

was added dropwise and stirred for 10 min. NaN₃(1.1×, 3.01 mmol,

196 mg, 65.01 MW) dissolved in cold H₂O (2 mL) was added

dropwise. After 1 h, the reaction was poured into water (40 mL),

which was extracted 3 times with CH₂Cl₂(40 mL each). The extracts

were washed with sodium bicarbonate and brine (100 mL each), dried

with sodium sulfate, filtered, and concentrated. Purification by silica

flash chromatography (100% CH₂Cl₂) yielded the azide as a light

brown solid (310 mg, 66%). ¹H NMR (600 MHz, d₆-DMSO): δ

11.025 (s, 1H, N−H), 7.295−7.281 (d, J = 8.4, 1H, indole-7H),

7.131−7.127 (d, J = 2.4, 1H, indole-4H), 6.737−6.719 (dd, J₁= 8.4, J₂

= 2.4, 1H, indole-6H), 6.108−6.106 (d, J = 1.2, 1H, indole-3H), 2.369

(s, 1H, methyl). ¹³C NMR (150 MHz, d₆-DMSO): δ 137.5, 133.9,

130.0, 129.5, 111.7, 111.6, 108.5, 99.0, 13.4. Melting point: 57−60 °C.

TLC (in 1:1 ethyl acetate:hexanes) R_f= 0.56. IR (film): 3409 cm⁻¹,

2111 (aryl-azide). Elemental analysis calculated for C₉H₈N₄: C, 62.78;

H, 4.68; N, 32.54. Found: C, 62.95; H, 4.68; N, 32.53.

1

mg of yellow solid. A crude H NMR showed a 1:3.5 ratio of product

to aldehyde. The filtrate was concentrated and added to the product/

aldehyde mixture, and 0.8 equiv of both 4-acetyl-pyridine (17 μL, 0.15

mmol) and piperidine (15 μL, 0.15 mmol) were added to the reaction.

This was refluxed for another 24 h, after which the reaction was

cooled, filtered, and rinsed with cold methanol, providing 9 mg (13%)

of pure, yellow product. ¹H NMR (600 MHz, d₆-DMSO): δ 12.397 (s,

1H, N−H), 8.815−8.805 (m, 2H, pyr-2.6H), 8.321 (s, 1H, indole-4H),

8.081−8.056 (d, J = 15.0, 1H, CCH), 7.820−7.808 (d, J = 7.2, 2H,

phenyl-2,6H), 7.761−7.728 (m, 4H, phenyl-4H, indole-6H, pyr-3,5H),

7.644−7.619 (t, J = 7.5, 2H, phenyl-3,5H), 7.587−7.573 (d, J = 8.4,

1H, indole-7H), 7.347−7.321 (d, J = 15.6, 1H, CCH), 2.639 (s, 3H,

methyl). ¹³C NMR (150 MHz, d₆-DMSO): δ 195.7, 187.9, 150.6,

146.9, 144.7, 138.8, 138.5, 138.4, 131.9, 130.1, 129.4, 128.4, 125.2,

123.9, 123.6, 121.1, 114.6, 111.9, 110.1, 12.1. Melting point: 258−262

°C. TLC (ethyl acetate:hexanes 4:1) R_f= 0.24. Elemental analysis

calculated for C₂₄H₁₈N₂O₂·0.65H₂O: C, 76.24; H, 5.14; N, 7.41; C,

75.96; H, 4.75; N, 7.16.

trans-3-(6-Benzoyl-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-

propen-1-one (31). In a dried 100 mL round-bottom flask under

argon, 29 (50 mg, 0.19 mmol) was dissolved in anhydrous methanol

(3 mL). 4-Acetyl-pyridine (21 μL, 0.19 mmol) and piperidine (19 μL,

0.19 mmol) were added, and the reaction was refluxed. After 24 h, the

reaction was cooled, filtered, and rinsed with cold hexanes and dried

1

under vacuum, yielding 52 mg (74%) of pure yellow solid. H NMR

(600 MHz, d₆-DMSO): δ 12.350 (s, 1H, N−H), 8.832−8.222 (d, J =

6.0, 2H, pyr-2,6H), 8.255−8.241 (d, J = 8.4, 1H, indole-4H), 8.125−

8.099 (d, J = 15.6, 1H, CCH), 8.008−7.999 (d, J = 5.4, 2H, pyr-

3,5H), 7.801 (s, 1H, indole-7H), 7.766−7.754 (d, J = 7.2, 2 H, phenyl-

2,6H), 7.683−7.642 (m, 2H, phenyl-4H, indole-5H), 7.585−7.563 (m,

3H, CCH, phenyl-3,5H), 2.659 (s, 3H, methyl). ¹³C NMR (150

MHz, d₆-DMSO): δ 195.5, 188.2, 150.6, 148.5, 144.6, 138.7, 138.2,

2-Methyl-3-carboxaldehyde-5-azido-1H-indole (35). In an oven-

dried, 100 mL round-bottom flask purged with argon, POCl₃(1.5×,

3.14 mmol, 291 μL) was added to anhydrous DMF (2.0 mL) at 0 °C.

Compound 34 (dissolved in DMF, 2 mL) was added dropwise. The

stirred reaction was slowly warmed to room temperature (RT). NaOH

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(1 N, 25 mL) and water (25 mL) were added, forming a precipitate

that was filtered and rinsed with cold water. The resulting white solid

was dried under vacuum, affording 369 mg (88%). H NMR (600

2-Methyl-5-methoxy-6-azido-1H-indole (39). In a dried, 100 mL

round-bottom flask under argon, 38 (227 mg, 1.29 mmol) was

dissolved in 90% AcOH (10 mL) and placed at 0 °C. The flask was

covered with foil, and the reaction was conducted in low light. NaNO₂

(98 mg, 1.43 mmol, 1.1 equiv) dissolved in H₂O (1 mL) was added

dropwise, and the mixture was stirred for 10 min. NaN₃(92 mg, 1.43

mmol, 1.1 equiv) dissolved in H₂O (1 mL) was added dropwise. After

45 min, the mixture was slowly poured into H₂O (25 mL) and

saturated K₂CO₃(19 mL) to form a neutral pH. This was extracted

with ethyl acetate (4 × 50 mL), washed with brine, dried with Na₂SO₄,

filtered, and concentrated. The crude product was purified by column

chromatography (ethyl acetate:hexanes 1:4), yielding 139 mg of pure

oil (53%). ¹H NMR (600 MHz, CDCl₃): δ 7.782 (s, 1H, N−H), 7.007

(s, 1H), 6.883 (s, 1H), 6.145 (s, 1H, indole-3H), 3.890 (s, 3H, o-

methyl), 2.388 (s, 3H, c-methyl). ¹³C NMR (150 MHz, CDCl₃): δ

147.0, 136.1, 130.7, 126.5, 123.1, 102.4, 102.0, 100.4, 56.5, 13.7. TLC

(in 1:1 ethyl acetate:hexanes) R_f= 0.60.

1

MHz, d₆-DMSO): δ 12.078 (s, 1H, N−H), 10.031 (s, 1H, CHO),

7.754−7.751 (d, J = 1.8, 1H, indole-4H), 7.423−7.409 (d, J = 8.4, 1H,

indole-7H), 6.913−6.896 (dd, J₁= 8.4, J₂= 1.8, 1H, indole-6H), 2.676

(s, 3H, methyl). ¹³C NMR (150 MHz, d₆-DMSO): δ 184.3, 149.7,

133.4, 133.0, 126.6, 114.4, 113.4, 112.8, 109.4, 11.5. Melting point:

degradation ca. 150 °C. TLC (in 1:1 ethyl acetate:hexanes) R_f= 0.19.

Elemental analysis calculated for C₁₀H₈N₄O: C, 59.99; H, 4.03; N,

27.99. Found: C, 60.03; H, 4.07; N, 27.96.

trans-3-(5-Azido-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-

propen-1-one (36). In a dried, 100 mL round-bottom flask under

argon and protected from light, 35 (80 mg, 0.40 mmol) was partially

dissolved in anhydrous methanol (5 mL). 4-Acetyl-pyridine (1.5 equiv,

66 μL, 0.60 mmol) and piperidine (40 μL, 0.40 mmol) were added,

and the reaction was stirred at 40 °C. After 24 h, the reaction was

cooled to RT, and the yellow precipitate was isolated via filtration and

rinsed with cold methanol. A crude ¹H NMR of this product showed a

ca. 1:3 ratio of product to the indole starting material. This crude

product was redissolved in anhydrous methanol (5 mL) along with the

concentrated filtrate, and another equivalent of 4-acetyl-pyridine (44

μL, 2.5 equiv total) and 5 equiv of piperidine (200 μL, 6 equiv total)

were added. The stirred reaction was heated at 40 °C. After 12 h, a

yellow precipitate was filtered and rinsed with cold methanol and dried

under vacuum, affording 47 mg (39%) of pure product. ¹H NMR (600

MHz, d₆-DMSO): δ 12.105 (s, 1H, N−H), 8.816−8.806 (dd, J₁= 4.8,

J₂= 1.8, 2H, pyr-2,6H), 8.066−8.041 (d, J = 15, 1H, CCH), 7.956−

7.945 (dd, J₁= 4.8, J₂= 1.8, 2H, pyr-3,5H), 7.661−7.657 (d, J = 2.4,

1H, indole-4H), 7.455−7.441 (d, J = 8.4, 1H, indole-7H), 7.401−

7.375 (d, J = 15.6, 1H, CCH), 7.001−6.984 (dd, J₁= 8.4, J₂= 1.8,

indole-6H), 2.587 (s, 3H, methyl). ¹³C NMR (150 MHz, d₆-DMSO):

δ 188.2, 150.6, 146.5, 144.9, 139.0, 133.9, 132.9, 126.8, 121.5, 113.9,

113.8, 113.0, 110.3, 109.2, 12.2. Melting point: decomposes ca. 190

°C. TLC (in 4:1 ethyl acetate:hexanes) R_f= 0.21. Elemental analysis

calculated for C₁₇H₁₃N₅O: C, 67.32; H, 4.32; N, 23.09. Found: C,

67.37; H, 4.33; N, 23.12.

2-Methyl-3-carboxaldehyde-5-methoxy-6-azido-1H-indole (40).

In an oven-dried, 100 mL round-bottom flask, purged with argon

and protected from light with foil, POCl₃(84 μL, 0.90 mmol, 1.5

equiv) was added to anhydrous DMF (1.0 mL) at 0 °C and stirred for

5 min. Next, 39 (dissolved in DMF, 1 mL) was added dropwise. The

stirred reaction was slowly warmed to RT. NaOH (1 N, 10 mL) and

water (10 mL) were added, and this was extracted with CH₂Cl₂(3 ×

20 mL). Extracts were washed with brine, dried with Na₂SO₄, filtered,

concentrated, and dried overnight under vacuum, producing 95 mg of

1

pure product (69%). H NMR (600 MHz, d₆-DMSO): δ 11.865 (s,

1H, N−H), 10.004 (s, 1H, CHO), 7.645 (s, 1H, indole-4H), 7.032 (s,

1H, indole-7H), 3.857 (s, 3H, o-methyl), 2.645 (s, 3H, c-methyl). ¹³C

NMR (150 MHz, d₆-DMSO): δ 184.8, 149.5, 149.4, 130.3, 124.1,

124.0, 114.4, 104.2, 103.5, 56.9, 12.2. TLC (in 1:1 ethyl

acetate:hexanes) R_f= 0.12.

trans-3-(6-Azido-5-methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyri-

dinyl)-2-propen-1-one (41). In a dried, 100 mL round-bottom flask

under argon and protected from light, 40 (70 mg, 0.30 mmol) was

partially dissolved in anhydrous methanol (5 mL). 4-Acetyl-pyridine

(50 μL, 0.45 mmol, 1.5 equiv) and piperidine (148 μL, 1.5 mmol, 5

equiv) were added, and the reaction was stirred at 40 °C. After 7 h, the

reaction was cooled to RT, and the precipitate was isolated via

2-Methyl-5-methoxy-6-nitro-1H-indole (37). In a 100 mL round-

bottom flask, 2-methyl-5-methoxyindole (370 mg, 2.30 mmol) was

dissolved in H₂SO₄(4 mL) after vigorous stirring, then placed at 0 °C.

In a separate flask, 1.87 g of NaNO₃(214 mg, 2.52 mmol) was

dissolved in H₂SO₄(4 mL), also after vigorous stirring, chilled to 0 °C,

and added dropwise to the indole. After addition, the reaction was

stirred for another 10 min and then poured into ice−water (200 mL),

precipitating a yellow product, which was extracted with ethyl acetate

(3 × 100 mL). Extracts were washed with saturated NaHCO₃and

brine, dried with Na₂SO₄, filtered, and concentrated. Purification by

column chromatography (ethyl acetate:hexane 2:3 → 3:2) yielded 296

mg of pure product (62%) (followed by 73 mg of 4-nitrated product,

1

filtration and rinsed with cold methanol. A crude H NMR of this

product showed a mixture of aldehyde with trace product. This

mixture was redissolved in the filtrate, more 4-acetyl-pyridine (165 μL,

5 equiv) and piperidine (148 μL, 5 equiv) were added, and the mixture

was set to react at 40 °C. After 48 h, although starting materials are still

seen on TLC, the crude reaction mixture was dry loaded onto silica

and purified by column chromatography (ethyl acetate:hexane 3:1),

eluting first the aldehyde, followed by the ketone, and finally the

1

product as a yellow solid (39 mg, 39%). H NMR (600 MHz, d₆-

DMSO): δ 11.784 (s, 1H, N−H), 8.815−8.805 (dd, J₁= 4.2, J₂= 1.8,

2H, pyr-2,6H), 8.073−8.047 (d, J = 15.6, 1H, CCH), 7.957−7.947

(dd, J₁= 4.2, J₂= 1.8, 2H, pyr-3,5H), 7.538 (s, 1H, indole-4H), 7.410−

7.384 (d, J = 15.6, 1H, indole CCH), 7.051 (s, 1H, indole-7H),

3.982 (s, 3H, o-methyl), 2.569 (s, 3H, c-methyl). ¹³C NMR (150

MHz, d₆-DMSO): δ 188.2, 150.6, 148.5, 145.6, 145.0, 139.1, 130.6,

123.6, 123.3, 121.5, 113.5, 109.5, 103.8, 103.7, 56.9, 12.3. Melting

point: degrades to black substance ca. 160 °C. TLC (in 4:1 ethyl

acetate:hexanes) R_f= 0.15. Elemental analysis calculated for

C₁₈H₁₅N₅O₂·C₄H₈O₂(trace ethyl acetate): C, 64.72; H, 4.60; N,

20.74. Found: C, 65.11; H, 4.71; N, 20.39.

Library Compounds. Compounds 1−8 used for initial screening

of methuosis-inducing activity (Figure 1) were obtained from

Hit2Lead.com, a division of Chembridge Corp. The identification

numbers of the compounds were as follows: 1, 5224450; 2, 5224466;

3, 5312531; 4, 7995005; 5, 7916760; 6, 6161388; 7, 5267766; and 8,

6155359. All compounds are certified by the vendor to be at least 90%

pure with NMR confirmation of structure.

1

15%; 4:1 regioselectivity). H NMR (600 MHz, CDCl₃): δ 8.350 (s,

1H, N−H), 7.983 (s, 1H, indole-7H), 7.066 (s, 1H, indole-4H), 6.211

(s, 1H, indole-3H), 3.958 (s, 3H, o-methyl), 2.477 (s, 3H, c-methyl).

¹³C NMR (150 MHz, CDCl₃): δ 148.7, 142.8, 134.7, 134.2, 129.1,

109.2, 102.7, 101.3, 57.1, 14.2. Melting point: 134−138 °C. TLC (in

1:1 ethyl acetate:hexanes) R_f= 0.37 (4-nitro product R_f= 0.25).

Elemental analysis calculated for C₁₀H₁₀N₂O₃·0.1C₆H₁₄: C, 59.27; H,

5.35; N, 13.04. Found: C, 59.33; H, 5.10; N, 12.98.

2-Methyl-5-methoxy-6-amino-1H-indole (38). In a hydrogenation

flask, 37 (270 mg, 1.31 mmol) was dissolved in 100% EtOH (35 mL),

and 10% Pd/C (40 mg) was added. The mixture was hydrogenated on

a Parr hydrogenator at 40 psi for 45 min. The red-pink solution was

filtered over Celite and rinsed with MeOH, concentrated, and dried

under vacuum, yielding 230 mg of pure product (99%). ¹H NMR (400

MHz, CDCl₃): δ 7.516 (s, 1H, N−H), 6.907 (s, 1H), 6.607 (s, 1H),

6.052 (s, 1H, indole-3H), 3.873 (s, 3H, o-methyl), 3.733 (bs, 2H,

NH₂), 2.365 (s, 3H, c-methyl). ¹³C NMR (150 MHz, d₆-DMSO): δ

142.8, 132.9, 131.5, 131.4, 119.5, 100.5, 98.7, 95.7, 55.6, 13.5 Melting

point: 144−148 °C. TLC (in 3:1 ethyl acetate:hexanes) R_f= 0.33.

Elemental analysis calculated for C₁₀H₁₀N₂O₃·0.1C₆H₁₄: C, 59.27; H,

5.35; N, 13.04. Found: C, 59.33; H, 5.10; N, 12.98.

Cell Culture. U251 human glioblastoma cells were purchased from

the DCT Tumor Repository (National Cancer Institute, Frederick,

MD). MCF-7 mammary carcinoma cells were obtained from The

American Type Culture Collection (Rockville, MD). TMZ-resistant

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Journal of Medicinal Chemistry

Article

U251 cells (U251-TR) were derived in our laboratory as described

previously.¹⁰MCF-7 cells selected for resistance to doxorubicin⁵⁶were

provided by Amadeo Parissenti, Northeastern Ontario Regional

Cancer Centre. Normal human skin fibroblasts were derived from a

skin biopsy as described previously.⁵⁷Unless stated otherwise, cell

lines were maintained in Dulbecco's modified Eagle's medium

(DMEM) with 10% (v/v) fetal bovine serum (FBS) (JR Scientific,

Woodland, CA) at 37 °C in an atmosphere of 5% CO₂/95% air.

Normal prestasis HMEC (specimen 184) were provided by Martha

Stampfer, Lawrence Berkeley Lab (Berkeley, CA). The HMECs were

maintained in M87A medium supplemented with cholera toxin and

oxytocin, essentially as described.⁵⁸

Cell Proliferation and Morphology. To generate cell growth

curves, U251 cells were plated in 35 mm dishes (100000 cells/dish)

and allowed to attach for 24 h. Thereafter, cells were treated with the

indicated compounds dissolved in DMSO or with vehicle alone. At

daily intervals, three parallel cultures were harvested from each group

by trypsinization, and aliquots of cell suspension were counted in a

Coulter Z1 particle counter. Phase-contrast images of live cells were

obtained using an Olympus IX70 inverted microscope equipped with a

digital camera and SPOT imaging software (Diagnostic Instruments,

Inc., Sterling Heights, MI).

Cell Viability. Cells were seeded in 96-well plates, with four

replicate wells for each culture condition. After addition of the

indicated concentrations of compounds, cell viability was determined

at a 48 h end point using a MTT-based assay as described.¹⁰

Absorbance at 570 nm was quantified on a SpectraMax Plus 384 plate

reader (Molecular Devices, Sunnyvale, CA).

Colony Formation. Cells were plated in 100 mm dishes at 2500

(U251 and U251-TR) or 1500 (MCF-7 and MCF-7 DoxR) cells per

dish. Beginning on the day after plating, the cells were exposed to the

indicated compounds for the periods of time noted in the figure

legends. The medium was then replaced (without compounds), and

cells were incubated for 10 days, with fresh medium added every 2

days. Colonies were visualized by washing with phosphate-buffered

normal saline, fixing for 10 min with ice-cold 100% methanol, and

staining with 1% (w/v) crystal violet (Acros Organics, Fisher

Scientific, Pittsburgh, PA) in 35% methanol. After 2−3 washes with

water, colonies containing at least 50 cells were counted using a

dissecting microscope or a Protocol 2 colony counter (Synbiosis,

Frederick, MD).

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ASSOCIATED CONTENT

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S

* Supporting Information

Spectral data for all synthesized compounds. This material is

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AUTHOR INFORMATION

■

Corresponding Author

*Tel: 419-530-2167. Fax: 419-530-1994. E-mail: paul.erhardt@

utoledo.edu (P.W.E.). Tel: 419-383-4161. Fax: 419-383-6228.

E-mail: william.maltese@utoledo.edu (W.A.M.).

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS

■

The work was supported by Grant R01 CA115495 (W.A.M.)

from the National Institutes of Health.

ABBREVIATIONS USED

■

DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DMF, dimethylfor-

mamide; DMSO, dimethylsulfoxide; GBM, glioblastoma multi-

forme; HMEC, human mammary epithelial cells; MTT, 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; RT,

room temperature; TMZ, Temozolomide

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Synthesis and evaluation of indole-based chalcones as inducers of methuosis, a novel type of nonapoptotic cell death

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