Methuosis is a form of nonapoptotic cell death characterized by an accumulation of macropinosome-derived vacuoles with eventual loss of membrane integrity. Small molecules inducing methuosis could offer significant advantages compared to more traditional anticancer drug therapies that typically rely on apoptosis. Herein we further define the effects of chemical substitutions at the 2- and 5-indolyl positions on our lead compound 3-(5-methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propene-1-one (MOMIPP). We have identified a number of compounds that induce methuosis at similar potencies, including an interesting analogue having a hydroxypropyl substituent at the 2-position. In addition, we have discovered that certain substitutions on the 2-indolyl position redirect the mode of cytotoxicity from methuosis to microtubule disruption. This switch in activity is associated with an increase in potency as large as 2 orders of magnitude. These compounds appear to represent a new class of potent microtubule-active anticancer agents.
Methuosis is a form of nonapoptotic cell death chn class="Chemical">aracterized by an accumulation of macropinosome-derived vacuoles with eventual loss of membrane integrity. Small molecules inducing methuosis could offer significant advantages compared to more traditional anticancer drug therapies that typically rely on apoptosis. Herein we further define the effects of chemical substitutions at the 2- and 5-indolyl positions on our lead compound 3-(5-methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propene-1-one (MOMIPP). We have identified a number of compounds that induce methuosis at similar potencies, including an interesting analogue having a hydroxypropyl substituent at the 2-position. In addition, we have discovered that certain substitutions on the 2-indolyl position redirect the mode of cytotoxicity from methuosis to microtubule disruption. This switch in activity is associated with an increase in potency as large as 2 orders of magnitude. These compounds appear to represent a new class of potent microtubule-active anticancer agents.
Conventional cancer
chemotherapy relies heavily on drugs that trigger
programmed cell death via activation of apoptosis pathways.[1,2] However, many n class="Disease">cancers harbor mutations in tumor suppressor genes
that function in apoptosis signaling.[3−5] Combined with increased
capacity for drug efflux and DNA repair, these mutations attenuate
apoptosis and contribute to the chemoresistance frequently encountered
in recurrent tumors.[6] Glioblastoma multiforme
(GBM) is the most common primary brain tumor in adults, and it is
a notorious example of cancer that is refractory to most forms of
chemotherapy. Treatment for GBM typically includes surgical excision,
followed by radiotherapy and administration of the DNA alkylating
agent temozolomide.[7] This standard-of-care
therapy marginally increases patient survival time, but no curative
treatment exists.[7,8] Identification of molecules that
can kill cancer cells via nonapoptotic cell death mechanisms may provide
inroads to circumvent the insensitivity of GBM and other cancers to
apoptosis. A number of nonapoptotic cell death pathways have been
defined,[9,10] and associated small molecules capable of
inducing these pathways have been reported.[11] Our group has focused on the development of compounds that can induce
a novel form of cell death termed methuosis.[12] First observed as a caspase-independent form of cell death triggered
by overexpression of activated Ras in GBM cells,[13,14] methuosis is characterized by the accumulation of large cytoplasmic
vacuoles derived from macropinosomes and nonclathrin-coated endosomes.
The vacuolated cells eventually undergo metabolic failure and lose
membrane integrity. In pursuit of small molecules that might be used
to induce methuosis in a therapeutic context, we initially identified
indolyl-pyridinyl-propenones (also referred to as indole-based chalcones)
that can trigger this form of cell death in GBM cells, including those
resistant to temozolomide.[15] Preliminary
structure–activity relationship (SAR) studies defined key requirements
for methuosis-inducing activity, resulting in a lead compound, 3-(5-methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propene-1-one, abbreviated
MOMIPP (1a, Figure 1), with activity
at low micromolar concentrations in cell-based assays.[16] The following features contributed to optimal
activity of the pharmacophore: (i) an indole-propenone-pyridine scaffold
where the pyridine’s attachment is in the para (4′)
position; (ii) a methoxy group at the 5-indolyl position; and, (iii)
small alkyl substitutions at the 2-indolyl position, i.e., R = Me
(1a) and Et (1b)[16,17] (Figure 1). More recent studies of longer-chain
aliphatic substitutions on the 2-indolyl position identified compounds
that retained the ability to induce cellular vacuolization but lost
the ability to kill GBM cells, as exemplified by compound 1c(17) (Figure 1).
These surprising findings suggest that induction of methuotic cell
death by specific indolyl-pyridinyl-propenones may involve pleiotropic
effects on cellular pathways beyond those responsible for perturbation
of macropinosome trafficking and vacuolization. Herein we evaluated
a new series of compounds with additional modifications at the 2-
and 5-indolyl positions. The results show that these compounds can
either maintain good methuosis-inducing activity or uncouple vacuolization
from cell death. An unexpected finding was that certain substitutions
at the 2-indolyl position conferred a substantially altered biological
profile, with disruption of microtubule polymerization becoming a
prominent feature. This was accompanied by a dramatic increase in
cytotoxicity. These compounds appear to represent a new class of potent
microtubule-active anticancer agents.
Figure 1
Structural requirements for optimal induction
of methuotic cell
death. Compound 1a is the current lead compound. The
ethyl analogue, 1b, is of similar potency. The propyl
derivative, 1c, induces cytoplasmic vacuolization, but
is not cytotoxic.[17]
Structural requirements for optimal induction
of methuotic cell
death. Compound 1a is the current lead compound. The
ethyl analogue, 1b, is of similn class="Chemical">ar potency. The propyl
derivative, 1c, induces cytoplasmic vacuolization, but
is not cytotoxic.[17]
Results
Chemistry
The 2- and 5-substitutedn class="Chemical">indolyl-pyridinyl-propenones
(Table 1, 2a–2q) were prepared by one of the following methodologies: (1) Regioselective
acylation from 4-substitutedN-BOC-protected 2-methylanilines
(6a–6d, 18) and Weinreb
amides[18] (7, 19) followed by acid-catalyzed cyclization and deprotections (Schemes 1 and 4) to afford 1H-indole
rings (8a–d, 20);[19] or (2) Synthetic manipulation at specific positions
on the formed indole ring to obtain 5-substituted amides (11, 12, 16 in Schemes 2 and 3), alkyl 2-indolylcarboxylates (23a–d in Scheme 5) or functionalized hydroxyl-based 2-indole intermediates (25, 30, 31 in Schemes 6 and 7). Synthesized indoles were then
formylated under Vilsmeier–Haack conditions and subsequently
condensed with 4-acetylpyridine producing final targets (2a–f, 2h, 2j–n, 2p, 2q) in a manner similar to
our previous reports.[16,17] Targets 2g, 2i, and 2o were prepared from their corresponding
indolyl-pyridinyl-propenone scaffolds.
Table 1
Summary of Growth
Inhibition Results
for Compounds Generated By Schemes 1–7a
(*) >10: Growth inhibition relative
to control did not reach 50% at the highest concentration tested (10
μM). (**) NI: No significant growth inhibition was detected
relative to vehicle control.
Scheme 1
Synthesis of 5-Alkyloxy-2-methylindolyl
Substituted Pyridinyl-Propenones 2a–d
Reagents and conditions: (a)
diethylsulfate in the case of 4a, or alkyl bromide, K2CO3, 2-butanone, reflux; (b) H2, Pd/C,
EtOAc; (c) di-tert-butyl dicarbonate, THF, reflux;
(d) 1. −40 to −50 °C, sec-butyllithium,
THF; 2. −50 to −10 °C, 7; 3. TFA/DCM (e) 1. POCl3, DMF; 2. 1 N NaOH; (f) 4-acetylpyridine, piperidine, MeOH,
reflux.
Scheme 4
Synthesis of 5-Methoxy-2-trifluoromethylindole-pyridinyl-propenone 2j
Reagents and conditions: (a)
1. THF, sec-butyllithium, −40 °C to −50
°C; 2. Weinreb amide 19, −50 °C to −10
°C; 3. TFA/DCM; (b) 1. POCl3, DMF; 2. 1 N NaOH; (c)
4-acetylpyridine, piperidine, MeOH.
Scheme 2
Synthesis of 5-Amino-2-methylindolyl-pyridinyl-propenone (2g) and 5-Substituted Amides 2e and 2f
Synthesis of 5-Alkyloxy-2-methylindolyl
Substituted Pyridinyl-Propenones 2a–d
Reagents and conditions: (a)
diethylsulfate in the case of 4a, or n class="Chemical">alkyl bromide, K2CO3, 2-butanone, reflux; (b) H2, Pd/C,
EtOAc; (c) di-tert-butyl dicarbonate, THF, reflux;
(d) 1. −40 to −50 °C, sec-butyllithium,
THF; 2. −50 to −10 °C, 7; 3. TFA/DCM (e) 1. POCl3, DMF; 2. 1 NNaOH; (f) 4-acetylpyridine, piperidine, MeOH,
reflux.
(*) >10: Growth inhibition relative
to control did not reach 50% at the highest concentration tested (10
μM). (**) NI: n class="Chemical">No significant growth inhibition was detected
relative to vehicle control.
Schemes 1–3 depict
the specific preparations of several 5-n class="Chemical">substituted analogues
designed to probe the effects of increasing the hydrocarbon chain
length in the ether linkage or replacing methoxy with nitrogen-containing
functionalities. The 5-alkyloxyindole analogues (Scheme 1) were synthesized from commercially available 3-methyl-4-nitrophenol
(3) by reaction with various alkyl-bromides, as in the
case of 4b–d, or diethylsulfate for 4a. Hydrogenation reduced the nitro-group to amines5a–5d, which were then protected with
BOC (6a–6d). Regioselective acylation
at the 2-methyl position was achieved using sec-butyllithium
and Weinreb amide 7. The resulting ketone intermediate
was cyclized and deprotected in a one-pot fashion utilizing excess
TFA to yield the 5-alkyloxy-2-methylindoles 8a–8d. Reaction of these indoles with POCl3 and DMF,
followed by treatment with 1 NNaOH, provided aldehydes 9a–9d which conveniently precipitated from the
reaction media. The aldehydes were condensed with 4-acetylpyridine
to provide the 5-substituted target compounds 2a–2d.
Analogues of 1a containing either amide
or n class="Chemical">amine substitution
at the 5-indolyl position were prepared as shown in Schemes 2 and 3. 5-Amino-2-methylindole
(10, Scheme 2) was prepared by
previously described methods[16] and used
to synthesize amide intermediates 11 and 12 by reaction with acetic anydride and di-tert-butyl
dicarbonate, respectively. Interestingly, compound 11 was isolated as the diacetylated amide under conditions of reflux
and excess reagent. The literature provides precedent for diacylation
to occur when aniline is subjected to similar conditions.[20] Conveniently, this readily isolable material
could be deployed directly in the next step because a single acetyl-group
was hydrolyzed during the NaOH-water workup leading to 13. Aldehydes 13 and 14 were condensed with
4-acetylpyridine to provide target compounds 2e and 2f. TFA deprotection of 2f in MeOH at rt yielded
the free amine probe 2g. The des-methyl
derivatives 2h and 2i (Scheme 3) were prepared from commercially available 15 in a fashion analogous to that for 2f and 2g but contain a methylene substitution between the amine and the indole
ring.
Synthesis of 5-Amino-2-methylindolyl-pyridinyl-propenone (2g) and 5-Substituted Amides 2e and 2f
Synthesis of 5-Aminomethylindolyl-pyridinyl-propenones 2h and 2i
Reagents
and conditions: (a)
di-tert-butyl dicarbonate, n class="Chemical">CH3CN, rt;
(b) 1. POCl3, DMF; 2. 1 NNaOH; (c) 4-acetylpyridine, piperidine,
MeOH, reflux; (d) TFA, MeOH, reflux.
Expanding
previous SAR studies of n class="Chemical">2-indolyl alkyl substitutions,[17] additional functionalized probes were prepared
as depicted in Schemes 4–7. Trifluoromethyl derivative 2j (Scheme 4), an isostere of 1a, was synthesized by acylating the 2-methyl substituent
on the N-BOC-protected aniline with trifluoromethylamide 19.[18,21] Excellent regioselectivity was
achieved by the dilithio species derived from 18 and sec-butyllithium in THF at −40 °C.[19] Cyclization and deprotection occurred in a one-pot
fashion with excess TFA affording 5-methoxy-2-trifluoromethylindole
(20). Synthesis of aldehyde 21 and condensation
to the final trifluoromethyl target 2j were accomplished
by our standard methods. Alkyl carboxylates (23a–d) were synthesized from commercially available acid 22 by Fisher esterification conditions using their corresponding
alcohols (Scheme 5). Formylation reactions to yield aldehydes 24a–24d, followed by Claisen-Schmidt condensation to provide 2k–2n, were conducted as described previously.[16,17] Interestingly, the isopropyl ester 2n appeared to be
the most sensitive to hydrolysis during workup and subsequent manipulation
or storage, likely owing to its better leaving group among this series.
Nevertheless, after saponification in aqueous base and MeOH, free
acid 2o was also readily obtained from 2k which was available in greater supply.
Synthesis of 5-Methoxy-2-trifluoromethylindole-pyridinyl-propenone 2j
Reagents and conditions: (a)
1. THF, n class="Chemical">sec-butyllithium, −40 °C to −50
°C; 2. Weinreb amide 19, −50 °C to −10
°C; 3. TFA/DCM; (b) 1. POCl3, DMF; 2. 1 NNaOH; (c)
4-acetylpyridine, piperidine, MeOH.
Synthesis
of the Analogous Series of Substituted Alkyl 2-Indolylcarboxylate-pyridinyl-propenones 2k–2n, as well as the Free Acid Analogue 2o
Reagents and conditions: (a)
HCl (g), n class="Chemical">R-OH, reflux; (b) 1. POCl3, DMF; 2. 1 NNaOH;
(c) 4-acetylpyridine, piperidine, R-OH, reflux; (d) 2k, MeOH, 1 NNaOH.
Derivatives substituted
with n class="Chemical">hydroxyl functionality were synthesized
based upon the results obtained in our previous and current SAR studies
for 2-substituted indole. Subtle chemical modifications at this position
can alter activity and appear to be sensitive to both steric and electronic
properties. Hydroxymethyl probe 2p was obtained according
to Scheme 6. Previously
prepared intermediate ethyl 5-methoxyindole-2-carboxylate (23b) was reduced by LAH to 25, which was then protected
by acetylating the primary hydroxyl group (26).[22] Conveniently, the acetyl group was then hydrolyzed
during the alkaline conditions used to quench the acidic reagents
generated during the Vilsmeier’s step that produce aldehyde 27. The aldehyde was condensed with 4-acetylpyridine to obtain 2p. This final target compound proved to be unstable when
stored at rt in DMSO for 48 h (see associated note in the Experimental Section), presumably due to the presence
of a benzylic alcohol conjugated to an α,β-unsaturated
ketone. To circumvent the inherent instability of 2p,
we also synthesized an analogue containing a hydroxypropyl functionality
using the methodology in Scheme 7. Alcohol 25 was oxidized to 5-methoxyindole-2-carboxaldehyde
(28) by MnO2 in EtOAc heated to reflux. The
aldehyde was subjected to Wittig conditions using phosphonium ylide
(Ph3PCHCO2C2H5) to produce
the α,β-unsaturated ester 29, which was reduced
to 2-hydroxypropylindole 30 using excess LAH. The primary
hydroxyl group was protected as the O-acetate (31) under
conditions of excess acetic anhydride, TEA, and CH3CN.
Standard reactions of formylation (32) and condensation
afforded hydroxypropyl analogue 2q. As anticipated, the
O-acetyl group was hydrolyzed under these conditions.
Synthesis
of 2-Hydroxymethyl-5-methoxyindole-pyridinyl-propenone 2p
Substitutions at the 5-Position of the Indole Ring
Cells experiencing methuosis initially undergo extreme cytoplasmic
vacuolization which can be readily assessed by phase contrast microscopy.
As viability is compromised (usually between 24 and 48 h), the cells
detach from the culture surface and lose membrane integrity. Previous
studies have established that the n class="Chemical">sulforhodamine B (SRB) colorimetric
assay, which measures protein associated with adherent cells, is useful
for evaluating the loss of viable cells and for ranking the relative
potency of methuosis-inducing compounds.[17] Although methuosis can occur in a broad spectrum of humantumor
cell lines,[12,14−16] our previous
SAR studies were carried out with U251glioblastoma cells. Therefore,
we continued to use this cell line in the present studies. Growth
inhibitory (GI) activities for all target compounds are recorded in
Table 1 as the dose able to achieve 50% inhibition
(GI50) compared to growth within control cultures treated
with only vehicle.
When 1a’s methoxy-group
is replaced by ethoxy (2a), isopropoxy (2c), or butoxy (2d) GI activity was mn class="Chemical">arkedly reduced.
In contrast, the GI50 value for the 5-propoxy compound
(2b) remained similar to that of 1a. As
shown in Figure 2, the morphology of cells
treated with 2.5 μM 1a was consistent with methuosis.
Robust cytoplasmic vacuolization was observed within 4 h. The vacuoles
persisted for the duration of the 48 h time-course with no increase
in cell density. By 48 h, some nonviable cells could be observed detaching
from the dish. Cytotoxicity was more evident at 10 μM, with
very few viable cells remaining on the dish. The effects of 2b at 2.5 μM were essentially the same as those observed
with 1a. In accord with its complete lack of GI activity,
the morphological effect of the butoxy derivative 2d contrasted
sharply with that of the propoxy 2b in that the former
produced much fewer and smaller vacuoles and had little effect on
cell density. Interestingly, the ethoxy (2a) and isopropoxy
(2c) derivatives exhibited intermediate activity, with
some induction of cellular vacuolization, but little or no inhibition
of cell growth. This apparent dissociation between vacuolization and
inhibition of cell growth/viability was similar to what we observed
previously with some of the 2-indolyl-substituted pyridinyl-propenones
upon lengthening the alkyl chain.[17]
Figure 2
Evaluation
of the impact of increasing the indolyl 5-alkyloxy chain
length on the morphological effects of 2-methylindolyl-pyridinyl-propenones
in U251 glioblastoma cells. Phase contrast images of live cells were
obtained as described in the Experimental Section. Compounds were added at the concentrations listed at the top of
each panel. The control cells shown in the top panel received an equivalent
volume of the DMSO vehicle.
Evaluation
of the impact of increasing the indolyl 5-alkyloxy chain
length on the morphological effects of n class="Chemical">2-methylindolyl-pyridinyl-propenones
in U251glioblastoma cells. Phase contrast images of live cells were
obtained as described in the Experimental Section. Compounds were added at the concentrations listed at the top of
each panel. The control cells shown in the top panel received an equivalent
volume of the DMSO vehicle.
The 5-amino derivative 2g had no GI activity
in the
concentration range tested (Table 1). It also
failed to induce vacuolization at 2.5 μM, although it did cause
vacuolization after 48 h at 10 μM (Figure 3). Likewise, neither the acetamide (2e), nor n class="Chemical">N-BOC (2f) analogues showed substantial growth
inhibition below 10 μM (Table 1), although
both compounds induced vacuolization when applied at 2.5 μM
and 10 μM (Figure 3). Thus, the activities
of 2e, 2f, and 2g were similar
to 2a and 2c, insofar as all of these compounds
triggered the accumulation of vacuoles without causing growth arrest
or cytotoxicity. The 2-position des-methyl versions
of the preceding N-BOC and free amine compounds,
with a methylene spacer between the nitrogen and the indole ring at
the 5-position (2h and 2i), gave mixed results.
Primary amine 2i was completely inactive in both growth
inhibition and vacuolization assays while the behavior of the N-BOC derivative 2h was somewhat anomalous.
It did not induce vacuolization or cell death (Figure 3) but caused a modest reduction in the growth rate of the
cells at concentrations above 2.5 μM (Table 1).
Figure 3
Evaluation of the impact of amine, acetamide, and N-BOC substitutions
at the 5-position of the indole ring on the morphological effects
of 2-methylindolyl-pyridinyl-propenones. Phase contrast images of
live cells were obtained as described in the Experimental
Section. Compounds were added at the concentrations listed
at the top of each panel.
Evaluation of the impact of amine, n class="Chemical">acetamide, and N-BOC substitutions
at the 5-position of the indole ring on the morphological effects
of 2-methylindolyl-pyridinyl-propenones. Phase contrast images of
live cells were obtained as described in the Experimental
Section. Compounds were added at the concentrations listed
at the top of each panel.
Substitutions at the 2-Position of the Indole Ring
To further explore the influence of the 2-position on the biological
activity of the pharmacophore, we n class="Chemical">substituted the methyl group of
the prototype 1a with a lipophilic CF3 group.
Surprisingly, the GI activity of this probe (2j) was
increased by nearly an order of magnitude compared with 1a (Table 1). As shown in Figure 4, the majority of the cells treated with 2j at
2.5 μM rounded up and detached by 24–48 h, and most of
the floating cells were determined to be nonviable by Trypan blue
dye exclusion. Similar morphological effects were observed at concentrations
as low as 0.6 μM. In cultures examined at 4 h, before the cells
began to detach from the surface, we observed the formation of small
cytoplasmic vacuoles and extensive blebbing of the plasma membrane
(Figure 4). This phenotype is quite distinct
from the extensive accumulation of large macropinocytotic vacuoles
that we have come to associate with the onset of methuosis (e.g.,
Figure 2, 1a and 2b). It suggests that the increased cytotoxicity of the 2-trifluoromethyl
derivative may be related to a different biological activity.
Figure 4
Evaluation
of the impact of different 2-indolyl substitutions on
the morphological effects of 5-methoyxyindolyl-pyridinyl-propenones.
Phase contrast images of live cells were obtained as described in
the Experimental Section. Compounds were added
at the concentrations listed at the top of each panel.
Evaluation
of the impact of different 2-indolyl substitutions on
the morphological effects of n class="Chemical">5-methoyxyindolyl-pyridinyl-propenones.
Phase contrast images of live cells were obtained as described in
the Experimental Section. Compounds were added
at the concentrations listed at the top of each panel.
In light of the preceding findings, we expanded
our compound library
to include other lipophilic-linked n class="Chemical">arrangements such as derivatives
containing alkyl 2-indolecarboxylates. As noted in Table 1, the methyl (2k), ethyl (2l), and n-propyl (2m) esters stood out
as being much more potent inhibitors of cell growth and viability
than any of the previously tested indolyl-pyridinyl-propenones, with
the GI50 of 2m approaching 10 nM (Table 1). Morphologically, the cells subjected to these
probes resembled those exposed to the trifluoromethyl derivative, 2j, with early membrane blebbing and cell rounding, followed
by extensive loss of adherent cells by 24 h (Figure 4). In the cases of the most potent ethyl and propyl esters
(2l and 2m), cytotoxic effects were observed
as low as 0.1 μM, where the less potent methyl ester (2k) and CF3 (2j) analogues were no
longer effective at this concentration. Interestingly, the GI activity
and morphological effects of the isopropyl ester (2n)
and the carboxylic acid (2o) were either markedly attenuated
or completely eliminated (Table 1, Figure 4). The striking influence of the nature of the aliphatic
chain on the biological activity of this series of alkyl carboxylates
underscores the previously reported sensitivity of the indolyl-pyridinyl-propenones
to synthetic manipulations at the 2-position of the indole ring,[17] although this time within the context of lipophilic
substituents that also appear to prompt a different mechanism.
A third type of 2-indolyl substitution is displayed by the hydroxymn class="Chemical">ethyl
(2p) and hydroxypropyl (2q) probes. Remarkably,
reduction of the ester reverted the biological activity of these compounds
to the methuosis phenotype, with GI values in the low micromolar range
typical of the other methuosis-inducing compounds (e.g., 1a and 2b, Table 1). Likewise,
cell morphology was characterized by extensive accumulation of large
cytoplasmic vacuoles, with loss of viable adherent cells at concentrations
≥ 2.5 μM.
Cell Cycle Effects
The striking increase in cytotoxicity
and the distinct morphological effects of the n class="Chemical">alkyl esters 2k–2m suggested that these compounds might be affecting
cell growth and viability via a mechanism distinct from the methuosis-inducing
compounds like 1a. Cell contraction, rounding, and detachment
from the substratum are typically observed in cultured cells treated
with mitotic inhibitors such as colchicine and vinblastine. Therefore,
we utilized flow cytometry to generate DNA histograms from cells treated
for 24 h with the two most potent agents, 2l and 2m. At a concentration of 3 μM, both compounds caused
a substantial accumulation of cells in the G2/M phase of the cell
cycle (Figure 5A). The apparent mitotic arrest
was accompanied by a significant increase in the percentage of cells
in the sub-G1/G0 compartment (Figure 5B), indicative
of cell death by mitotic catastrophe. Similar cell cycle effects of 2l and 2m were observed at 0.1 μM (Figure 5C), which represents the lower end of the concentration
range at which substantial morphological perturbations were observed
in Figure 4. Interestingly, the percentage
of cells in the G2/M phase actually was slightly higher in cultures
treated with 2m at 0.1 μM compared with 3 μM
(Figure 5B,C). This could be related to a higher
percentage of dead cells derived from the G2/M-arrested population
in the cultures treated with the higher concentration of 2m.
Figure 5
Effects
of selected indolyl-pyridinyl-propenones on cell cycle
distribution in U251 cells. (A) DNA histograms of cells treated with
the indicated compounds at 3 μM for 24 h were generated by flow
cytometry as described in the Experimental Section. (B) Cells were treated with each compound for 24 h and the percentage
of cells registering as having less than the G1/G0 DNA content (an
indication of non-viable cells) is depicted in the left panel. In
the same study the percentage of viable cells in each phase of the
cell cycle was determined after gating out the sub-G1/G0 counts (right
panel). (C) The assessment of cell death and cell cycle distribution
was repeated in U251 cells treated with compounds at 0.1 μM
instead of 3 μM. Values in B and C are means (± S.D.) derived
from three separate cultures. The G2/M-phase and sub-G0/G1 cell populations
in the cultures treated with 2l and 2m were
significantly increased compared to the DMSO control at all concentrations
(p ≤ 0.05).
Effects
of selected indolyl-pyridinyl-propenones on cell cycle
distribution in n class="CellLine">U251 cells. (A) DNA histograms of cells treated with
the indicated compounds at 3 μM for 24 h were generated by flow
cytometry as described in the Experimental Section. (B) Cells were treated with each compound for 24 h and the percentage
of cells registering as having less than the G1/G0 DNA content (an
indication of non-viable cells) is depicted in the left panel. In
the same study the percentage of viable cells in each phase of the
cell cycle was determined after gating out the sub-G1/G0 counts (right
panel). (C) The assessment of cell death and cell cycle distribution
was repeated in U251 cells treated with compounds at 0.1 μM
instead of 3 μM. Values in B and C are means (± S.D.) derived
from three separate cultures. The G2/M-phase and sub-G0/G1 cell populations
in the cultures treated with 2l and 2m were
significantly increased compared to the DMSO control at all concentrations
(p ≤ 0.05).
In contrast to the results with 2l and 2m, the cell cycle distribution of cells treated with methuosis-inducing
compounds such as 1a and 2b was similn class="Chemical">ar
to the DMSO-treated control (Figure 5). The
latter finding is consistent with our previous studies of cells undergoing
methuosis in response to overexpression of H-Ras (G12V),[13] wherein we observed that the cell cycle distribution
of the vacuolated cells did not change significantly prior to the
loss of viability. It should also be noted that the presence of normal
G1/G0 and S-phase populations in the cultures treated with 1a or 2b is not unexpected, since methuotic cell death
triggered by 1a typically takes longer than 24 h at drug
concentrations below 10 μM. In Figure 6, several additional compounds were tested for cell cycle effects
at a single concentration (3 μM). Arrest of U251 cells in the
G2/M phase, with a corresponding accumulation of sub-G1/G0 cells,
was clearly evident in cultures treated with the 2-trifluoromethyl
(2j) and methyl ester (2k) derivatives (Figure 6). The effects were very similar to those observed
with colcemid, a known microtubule disruptor (Figure 6). In contrast, free acid 2o had no detectable
effect on cell cycle distribution, reflecting its lack of activity
in the SRB and morphology assays.
Figure 6
Effects of additional indolyl-pyridinyl-propenones
on cell cycle
distribution in U251 cells. DNA histograms of cells treated with the
indicated compounds at 3 μM for 24 h were generated by flow
cytometry as described in the Experimental section. The results were analyzed to determine (A) the percentage of nonviable
cells with sub-G1/G0 DNA content and (B) the percentage of viable
cells in each phase of the cell cycle. Values are means (±S.D.)
derived from three separate cultures. The G2/M-phase and sub-G0/G1
cell populations in the cultures treated with 2j, 2k, and colcemid were significantly increased compared to
the DMSO control (p ≤ 0.05).
Effects of additional indolyl-pyridinyl-propenones
on cell cycle
distribution in n class="CellLine">U251 cells. DNA histograms of cells treated with the
indicated compounds at 3 μM for 24 h were generated by flow
cytometry as described in the Experimental section. The results were analyzed to determine (A) the percentage of nonviable
cells with sub-G1/G0 DNA content and (B) the percentage of viable
cells in each phase of the cell cycle. Values are means (±S.D.)
derived from three separate cultures. The G2/M-phase and sub-G0/G1
cell populations in the cultures treated with 2j, 2k, and colcemid were significantly increased compared to
the DMSO control (p ≤ 0.05).
Effects on Microtubule Polymerization
The apparent
n class="Disease">mitotic arrest of cells treated with 2j–2m prompted us to examine the effects of this class of compounds
on microtubule polymerization in intact cells. We began by assessing
the integrity of the microtubule network by immunofluorescence microscopy,
using an antibody against α-tubulin. As shown in Figure 7, the DMSO-treated control cells displayed a well-developed
array of microtubules radiating from the juxtanuclear microtubule
organizing center. In contrast, cells treated with 3 μM 2m had a tubulin staining pattern that was diffuse and disorganized.
At a lower concentration (0.1 μM), 2m had a similar
but somewhat less severe effect on microtubule organization. Cells
treated with the methuosis-inducer 1a appeared to contain
intact cable-like microtubules, although their organization was distorted
by the accumulation of large vacuoles throughout the cytoplasm.
Figure 7
Immunofluorescence
imaging of tubulin (red fluorescence) in cells
treated for 24 h with the methuosis-inducing compound 1a and the 2-indolyl propyl ester 2m. The nuclei are visualized
with DAPI (blue fluorescence).
Immunofluorescence
imaging of tubulin (red fluorescence) in cells
treated for 24 h with the methuosis-inducing compound 1a and the n class="Chemical">2-indolyl propyl ester 2m. The nuclei are visualized
with DAPI (blue fluorescence).
To obtain a more direct indication of the effects of the
compounds
on tubulin polymerization, we utilized an established biochemical
approach in which the drug-treated cells are lysed under conditions
designed to preserve the native polymerization state of the microtubules.
The polymerized tubulin is then n class="Gene">separated from soluble tubulin by
high-speed centrifugation and quantified by Western blot analysis.
The results, depicted in Figure 8, demonstrate
that the three most potent compounds, which caused mitotic arrest
in Figures 5 and 6 (2j, 2l, and 2m), produced substantial
declines in the relative amount of tubulin detected in the polymerized
fraction. Lowering the concentration from 3 μM to 0.1 μM
diminished but did not eliminate the effect on tubulin polymerization,
consistent with the immunofluorescence observations in Figure 7. The results were very similar to those obtained
with the positive control, colchicine, applied at the same concentrations.
The proportion of polymerized tubulin in cells treated with the methuosis-inducing
compounds, 1a and 2b, and the inactive derivative meta-MOMIPP,[17] was similar to
that detected in the DMSO control.
Figure 8
Effects of selected indolyl-pyridinyl-propenones
on tubulin polymerization
in cultured U251 cells. Cells were treated for 4 h with the indicated
compounds at a final concentration of 3 μM and then fractionated
under conditions designed to preserve the native polymerization state
of the microtubules. The percentage of polymerized versus soluble
tubulin was determined by immunoblot analysis as described in the Experimental Section. For each compound the fraction
of tubulin in the polymerized state was expressed as a percentage
of the polymerized tubulin determined in a parallel control culture
treated with DMSO alone. The results represent the mean (±S.D.)
of determinations from three separate experiments. The decreases in
the percentage of polymerized tubulin in cells treated with 2j, 2l, 2m and colchicine (at 3
μM) were significant at p ≤ 0.05.
Effects of selected indolyl-pyridinyl-propenones
on tubulin polymerization
in cultured n class="CellLine">U251 cells. Cells were treated for 4 h with the indicated
compounds at a final concentration of 3 μM and then fractionated
under conditions designed to preserve the native polymerization state
of the microtubules. The percentage of polymerized versus soluble
tubulin was determined by immunoblot analysis as described in the Experimental Section. For each compound the fraction
of tubulin in the polymerized state was expressed as a percentage
of the polymerized tubulin determined in a parallel control culture
treated with DMSO alone. The results represent the mean (±S.D.)
of determinations from three separate experiments. The decreases in
the percentage of polymerized tubulin in cells treated with 2j, 2l, 2m and colchicine (at 3
μM) were significant at p ≤ 0.05.
Discussion
The
indolyl-pyridinyl-propenones described in this report are related
to naturally occurring flavonoid precursors termed chalcones, which
consist of an α,β-unsaturated ketone linking two nonheterocyclic
aromatic rings. Over the past two decades a large number of naturally
occurring and synthetic chalcones and chalcone derivatives have been
characterized as having anticancer activities.[23−25] Our group has
focused on a unique class of such molecules wherein the aromatic ring
systems consist of indolyl- and pyridinyl-moieties. In particular,
we have established that certain members of this family, namely, 1a and its des-methoxy analogue (MIPP), can
induce methuosis and kill temozolomide-resistant glioma cells and
doxorubicin-resistant breast cancer cells at low micromolar concentrations.[15,16] The cytostatic and cytotoxic effects of 1a are moderately
selective for cancer cells compared with normal fibroblasts[16] (Supporting Information
Figure S1). Recently, others have described unrelated small
molecules (vacquinols) that also induce methuosis in cultured glioma
cells and suppress the growth of tumor xenografts in mouse models.[26] These studies have stimulated interest in evaluating
the therapeutic potential of methuosis-inducing compounds.Two
fundamental questions about the methuosis-inducing compounds
remain unresolved. The first relates to the identities of their protein
tn class="Chemical">argets. Previous studies suggested that induction of methuosis is
due to drug interaction with specific proteins, rather than general
covalent protein modification by Michael addition. In particular,
the reversibility of methuosis upon drug wash-out during the early
stages and the strict dependency of the biological effects on the para-configuration of the pyridinylnitrogen support this
concept.[16,17] While reassuring in terms of highlighting
specificity, the tendency of these compounds to be inactivated by
many modifications on the pyridinyl or indolyl moieties has made the
use of conventional affinity-based approaches for drug target identification
quite challenging.
A second key question concerns the mechanism(s)
leading to cell
death, particuln class="Chemical">arly the relationship between the formation of cytoplasmic
vacuoles and the ultimate loss of cell viability. Our initial SAR
studies with a directed library of compounds revealed that analogues
that failed to induce vacuolization generally failed to cause cell
death.[15,16] Thus, we inferred that vacuolization was
an important contributing factor in the cell death program. However,
our recent studies with certain aliphatic 2-indolyl substitutions,[17] coupled with the present observations with some
of the substituents at the indolyl 5-position (e.g., 2a, 2c, 2e, 2f), indicate that
vacuolization does not necessarily lead to growth arrest and cell
death in every case. Thus, our current working hypothesis is that
the cytotoxicity of certain indolyl-pyridinyl-propenones is due to
pleiotropic effects, combining perturbations of macropinosome/endosome
trafficking with alterations of cellular signaling or metabolic pathways
that remain to be defined.
In the present study we have identified
several new indolyl-pyridinyl-propenones
that exhibit robust biological activities. For the purpose of discussion,
we consider the active compounds as falling into three discernible
classes based on the distinct celluln class="Chemical">ar phenotypes that they induce
in cultured cells (Figure 9). The compounds
in Class 1 cause striking cellular vacuolization, but the cells continue
to proliferate and remain viable. Examples of Class 1 compounds are
drawn from this report (2a, 2c, 2e, 2f) and our previous publication.[17] We envision that the Class 1 compounds target proteins
involved in the formation of macropinosomes, the recycling of these
structures, or the fusion of macropinosomes with lysosomes. The complexity
of the phenotype suggests that there may be multiple Class 1 targets,
but we cannot exclude the possibility that perturbation of a single
enzyme, structural protein, or signaling molecule could be responsible.
We also identified several new compounds with substantial methuosis-inducing
activity (2b, 2p, 2q), comparable to that of our previously
characterized lead compound 1a. In the context of these
studies, we equate methuosis with the Class 2 phenotype. It is similar
to the Class 1 phenotype in terms of massive cellular vacuolization,
but in this case the cells detach and undergo metabolic failure, culminating
with rupture of the cell membrane (i.e., methuosis). We speculate
that in addition to interacting with Class 1 targets, the Class 2
compounds have affinity for a separate set of protein targets involved
in metabolic and/or signaling pathways essential for cell viability.
Through these combined effects, the Class 2 compounds trigger methuotic
cell death. The most striking observation from the present study is
that certain modifications at the 2-position of the indole ring increased
the cytotoxic activity of the compounds by 1 or 2 orders of magnitude
(e.g., 2j–2m). The increase in potency
was accompanied by notable changes in the phenotype observed in glioma
cells treated with these compounds, which we term Class 3. In contrast
to the typical vacuolated morphology seen in cells undergoing methuosis,
the cells treated with the 2-trifluoromethylindole and the alkyl 2-indolylcarboxylate
esters underwent a rapid transition from early formation of small
vacuoles to very active membrane blebbing and cell contraction. This
was followed by microtubule depolymerization, mitotic arrest, and
massive detachment of cells from the substratum. These changes lead
us to conclude that compounds 2j–2m acquired the capacity to interact with one or more protein targets
involved in microtubule assembly or maintenance (Class 3 targets).
The early hint of vacuolization in cells treated with these compounds
suggests that macropinocytosis may also have been affected, possibly
via Class 1 or Class 2 targets. However, with the superimposition
of potent microtubule-destabilizing activity, disruption of the cytoskeleton
and consequent detachment of the cells occur before the Class 1 or
Class 2 phenotypes can develop.
Figure 9
Classification of biologically active
idolyl-pyridinyl-propenones
based on distinct cellular phenotypes elicited by modifications at
R1 and R2. In the hypothetical model, Class
1 compounds are envisioned as interacting with one or more Class 1
protein targets to induce perturbations in macropinosome trafficking,
resulting in cellular vacuolization without impairment of cell proliferation
or viability. Class 2 compounds interact with the same Class 1 targets,
but acquire the ability to bind additional Class 2 protein targets
that function in metabolic or pro-survival signaling pathways. The
combination of effects on Class 1 and Class 2 proteins results in
a distinct phenotype characterized by extreme vacuolization and cell
death via methuosis. The novel compounds in Class 3 gain the ability
to inhibit proteins involved in microtubule assembly in a concentration
range where Class 1 or 2 compounds have no effect on tubulin polymerization.
Hence, mitotic arrest, cell rounding, and death (presumably by mitotic
catastrophe) predominate as the main features of the Class 3 phenotype.
It is unclear whether the Class 3 compounds retain the capacity to
interact with Class 1 and 2 targets, although the early detection
of vacuoles prior to microtubule disruption and cell rounding suggests
that there may be some overlap.
Classification of biologically active
idolyl-pyridinyl-propenones
based on distinct celluln class="Chemical">ar phenotypes elicited by modifications at
R1 and R2. In the hypothetical model, Class
1 compounds are envisioned as interacting with one or more Class 1
protein targets to induce perturbations in macropinosome trafficking,
resulting in cellular vacuolization without impairment of cell proliferation
or viability. Class 2 compounds interact with the same Class 1 targets,
but acquire the ability to bind additional Class 2 protein targets
that function in metabolic or pro-survival signaling pathways. The
combination of effects on Class 1 and Class 2 proteins results in
a distinct phenotype characterized by extreme vacuolization and cell
death via methuosis. The novel compounds in Class 3 gain the ability
to inhibit proteins involved in microtubule assembly in a concentration
range where Class 1 or 2 compounds have no effect on tubulin polymerization.
Hence, mitotic arrest, cell rounding, and death (presumably by mitotic
catastrophe) predominate as the main features of the Class 3 phenotype.
It is unclear whether the Class 3 compounds retain the capacity to
interact with Class 1 and 2 targets, although the early detection
of vacuoles prior to microtubule disruption and cell rounding suggests
that there may be some overlap.
In terms of summarizing specific Sn class="Chemical">AR features that might
be gleaned
from the assembled compounds, several interesting points can be raised.
First, it appears that the distinctive microtubule-related actions
described above for the trifluoromethyl and ester analogues 2j–2m are prompted by the presence of
an electron withdrawing substituent placed at the indolyl 2-position.
In 1a the 2-methyl group is modestly electron donating
and the biological profile is that of the Class 2 phenotype. Substitution
with an electron withdrawing trifluoromethyl group (2j) redirects the profile to that of Class 3 and substitution with
electron withdrawing carbonyl systems such as those present in esters 2l and 2m prompts Class 3 with even stronger
potency. Alternatively, when the ester substituent is branched (2n) or is removed entirely so as to expose a carboxylic acid
moiety (2o), activity is significantly reduced or lost
completely. This is similar to our previous findings where exploration
of this vicinity with various alkyl substituents suggested that there
may be a pocket in 1a’s methuosis-related protein
targets that prefers to accommodate a methyl or ethyl group.[17] For that series, larger groups at the indolyl
2-position still caused vacuolization, but the cells did not proceed
to methuotic death. Indeed, it was our suspicion about this pocket
that led to the design of the more lipophilic 2j in order
to further probe the importance of this feature for interaction with
Class 2 targets. However, in addition to providing the desired increase
in lipophilicity relative to 1a, the CF3 substitution
also confers a significant electron withdrawing effect. Consistent
with the results from the esters 2l and 2m, the latter appears to induce a shift in the biological activity
of 2j from a Class 2 to Class 3 phenotype.
Contributing
to construction of a topological map near the n class="Chemical">indolyl
2-position, relative to interactions with methuosis-related protein
targets, are the unanticipated results for the two alcohols 2p and 2q. They are not complicated by having
additional electron withdrawing effects and, as expected, do not evoke
mitotic arrest and cell death by interaction with microtubules (Class
3). Instead, these substituents endow strong Class 2 effects even
though they are less lipophilic than their corresponding alkyl systems.
Furthermore, the high potency exhibited by the n-propyl
alcohol 2q does not correspond to the previously limited
size for simple alkyl arrangements wherein methyl or ethyl were preferred
for methuosis activity.[17] Indeed, the preference
for a propyl in this case resembles the SAR for the Class 3 family
of microtubule-interacting esters wherein the best alcohol adduct
was also n-propyl. This could suggest that there
are some commonalities among the protein targets engaged by the Class
2 and Class 3 indolyl-pyridinyl-propenones. At this point, for the
specific methuosis-related proteins, we speculate that the topology
of the putative binding pocket for the indolyl’s 2-position
may approximate a narrow groove that can accommodate simple (nonbranched)
alkyl groups that are lipophilic at the proximal end and capable of
hydrogen bonding at the distal end, provided that ionizable moieties
are not present anywhere along the alkyl chain.
Preferences
for the topology around the n class="Chemical">indolyl 5-position appear
to be more stringent, but the overall SAR is equally intriguing. Similar
to our former study[17] where the indolyl
2-position was probed with various alkyl groups, many of the substituents
placed at the 5-position were able to retain Class 1 activity, but
only a few demonstrated Class 2 methuosis activity. As shown previously,[16] a phenolic substitution is not active, while
a methoxy, as is present in 1a, prompts significant methuosis.
Such activity is greatly reduced by ethoxy, is returned by n-propoxy, and then is greatly reduced again by branched
alkyl or n-butoxy (2a, 2b, 2c, and 2d, respectively). Assuming that
a model similar to that postulated for the indolyl 2-position also
pertains to the indolyl 5-position, one might ask why a binding pocket
that accommodates methoxy would optimally accept an n-propoxy substituent, but not an ethoxy or butoxy group. In this
regard, it is worth noting that similar anomalies have been reported
for well-known systems that have undergone extensive SAR studies.
In one example, the “Goldilocks” nature[27] of an ethyl group inserted between a bulky aryl-system
and a simple methyl estercarbonyl moiety (thus constituting a 3-carbon
system) was exploited as being “just right” to allow
ready access for attack of β-adrenergic antagonists by esterases.
While the 3-carbon system endowed an ultrashort duration of action,
similar effects were not afforded by methyl (2-carbon system) or n-propyl (4-carbon system) spacers.[28,29] In this case the author speculated that the additional conformational
freedom afforded by the n-propyl group could cause
the ester to become tucked back toward other bulky features present
within the overall molecular framework. A second example of preferred
conformational arrangements comes from the literature on dopamine
receptor agonists. In this case, dopamine loses significant activity
when its primary amine becomes substituted with a broad range of alkyl
functionalities except for a distinct disubstitution situation wherein
at least one of the substituents is an n-propyl group.[30−32] In this heavily studied research arena, this remarkable effect is
referred to as the “n-propyl phenomenon”,
and it has prompted speculation that the dopamine receptor’s
ligand-binding pocket maintains a “unique geometry to accommodate
[just] an n-propyl group”. Our prior computational
studies on substitutedindolyl-pyridinyl-propenones suggest that their
side-chain substitutions remain quite flexible, with only subtle differences
in energy across several conformational possibilities for each increment
of added carbon atoms to the 2-position.[17] Thus, in our case, various arrangements being uniquely adopted by
only certain homologues during interaction with a protein surface
cannot be ruled out.
Further insight regarding the possible
topography n class="Chemical">around the indolyl
5-position comes from our finding that an ionizable free amine (2g, 2i), a neutral amide (2e), and
a pair of carboxamides having hydrogen bonding capabilities (2f, 2h) are all unable to induce methuosis. Although
weak GI activity was observed in the specific case where a BOC group
was synthesized on an amine spaced one methylene unit away from the
indolyl moiety (2h), this activity did not match the
Class 2 or 3 cytotoxic phenotypes. Thus, similar to the model for
the indolyl 2-position, we speculate that the protein region interacting
with the 5-position also may have a restrictive groove that can accommodate
a methoxy group or the special case of a n-propoxy
group, with the provisos that the latter must remain lipophilic throughout,
and that additional substituents capable of hydrogen bonding or ionizationare excluded anywhere along the alkyl chain.
In conclusion for
our SAR, we find it quite remn class="Chemical">arkable that such
profound changes in biological profiles can be unmasked around the
indolyl-pyridinyl-propenone structural motif with subtle manipulations
at either of just two positions. While we offer proposals for what
the topography may look like in the relevant binding domains of the
unknown Class 2 protein targets, the assumptions are based upon the
present state of SAR with a limited spectrum of structural probes.
Nevertheless, until specific proteins are identified, these early
models can serve as useful conceptual tools for the design of new
probes to map the structural determinants responsible for the distinct
biological activities of the indolyl-pyridinyl-propenones.
Numerous
n class="Chemical">chalcones and chalcone-derivatives displaying antimitotic
activity have been characterized previously.[24,33,34] While many of these compounds were identified
by screening collections of synthetic chalcones in cell-based proliferation
assays,[35,36] others emerged from molecular modeling and
design studies aimed at creating new structures that might compete
for tubulin sites known to bind established microtubule inhibitors
(e.g., colchicine, combretastatins).[37] Most recently, efforts to develop curcumin analogues that
might kill cancer cells by targeting the NFκB activation pathway
serendipitously yielded a series of potent ortho-aryl
substitutedchalcones with the ability to bind to the colchicine site
on tubulin and induce mitotic arrest and apoptotic cell death.[38] Because of their distinct structural profiles
and potencies, the trifluoromethyl and ester analogues described herein
appear to represent a new class of potent microtubule-active anticancer
compounds.
Among the various traditional n class="Chemical">chalcones investigated
as potential
anticancer agents, indole-based chalcones have received comparatively
little attention until recently. In addition to the methuosis-inducing
indolyl-pyridinyl-propenones described by our group,[15−17] the other class of antiproliferative/cytotoxic indolyl-chalcones
comprises the 1-(N-methylindolyl)-3-phenylpropenones.[39−41] SAR studies of analogues with different substituents on the phenyl
ring revealed that 3-(1-methyl-1H-indol-3-yl)-1-(2,4,6-trimethoxyphenyl)-2-propen-1-one, 33 (JAI-51)[41−43] produced mitotic arrest at low micromolar concentrations.
This was attributed to direct inhibition of tubulin polymerization
based on the results of cell-free tubulin assembly assays.[43] Although it is possible that our 2-trifluoromethylindole
and ester derivatives might operate in a similar way to disrupt microtubules,
we have not yet obtained definitive evidence to discriminate between
direct tubulin binding and other indirect mechanisms, such as interference
with microtubule-associated proteins. The possibility that the mechanism
might differ from that described for 33 is suggested
by the much higher potency and unique structural features of the indolyl-pyridinyl-propenones.
In particular, our Class 3 microtubule-disrupting compounds lack the N-methyl group on the indole ring found in 33 and other antimitotic 1-(N-methylindolyl)-3-phenylpropenones.
Another key difference between our current set of compounds and previously
reported antimitotic chalcones relates to the pyridinyl moiety which
is uniquely present in our scaffold and requisite for optimal activity.
Specifically, cytotoxicity (via methuosis) was eliminated by switching
the position of the pyridinylnitrogen from para-
to meta-.[17] In a preliminary
study, we have determined that altering the position of the pyridinylnitrogen from para- to meta- in
the context of 2l and 2m, has a similar
negative influence on the antiproliferative activity of these microtubule-disrupting
compounds, raising their GI50’s by approximately
2 orders of magnitude (Supporting Information
Figure S2).
Drugs that affect microtubule dynamics are
among the most widely
employed antin class="Disease">cancer agents.[44,45] Distinct classes of
antimitotic compounds include the Vinca alkaloids
(e.g., vincristine, vinblastine) and colchicine analogues (e.g, combretastatins)
both of which are considered microtubule destabilizers, and the taxanes
(e.g., paclitaxel) which function as microtubule overstabilizing agents.[45,46] Although they are effective against many types of cancer, these
compounds are not without drawbacks in the clinic. Side effects, such
as peripheral neuropathy and neutropenia, may limit the doses and
treatment regiments that can be tolerated. Development of drug resistance
is also a factor that limits efficacy in many cases. The latter may
entail both induction of drug efflux pumps and alterations in the
intrinsic properties of the microtubules (e.g., tubulin isotypes or
posttranslational modifications).[47,48] Finally, the Vinca alkaloids[49] and taxanes[50] exhibit low permeability through the blood–brain
barrier, minimizing efficacy for treating primary or metastatic tumors
in the central nervous system. For these reasons, the identification
of new microtubule-directed agents with unique profiles continues
to be of interest. Indolyl-pyridinyl-propenonesare particularly intriguing
candidates for further study because published findings with other
indole-based chalcones suggest that they are poor substrates for drug
efflux pumps and are able to cross the blood–brain barrier.[43] Indeed, several functionalized chalcones have
been shown to actively impair drug transporters like P-glycoprotein
and breast cancer resistance protein.[43,51,52] It remains to be determined if this property extends
to the compounds described in this report. Our novel trifluoromethyl
and ester analogues could be particularly attractive prototypes for
future study, as they may operate through a combination of mechanisms
to induce features of both methuosis (disruption of macropinosome
trafficking) and microtubule destabilization. In this regard, it will
be important to discern if these compounds trigger cell death through
pathways similar to those promoted by conventional antitubulin compounds
(i.e., apoptosis, mitotic catastrophe)[53,54] or instead
operate through a novel cell death program. Ultimately, the potential
therapeutic utility of this class of compounds will depend on further
evaluation of cytotoxicity and selectivity in a broad panel of transformed
and nontransformed cell lines and determination of their pharmacokinetic
properties in vivo.
Experimental Section
General
Description
All reactions were performed in
oven-dried 2-neck round-bottom flasks under an atmosphere of either
Ar or n class="Chemical">N2 and stirred with Teflon-coated magnetic bars.
TLC (silica gel F254 plates, Baker-flex) was used to monitor
progress of all reactions with visualization performed under 254 nm
UV light. Reagent grade and anhydrous solvents were purchased from
Sigma-Aldrich and used without further purification unless otherwise
noted. Silica gel sorbent (230–400 mesh) was purchased from
Fisher Scientific. Samples to be purified by column chromatography
were dissolved in a minimal volume of solvent and then adsorbed onto
silica gel (5–10× the amount of sample by weight) by evaporation
under reduced pressure until the solid composite was free-flowing.
The dry loaded sample was then applied to the top of the prepacked
column bed allowing the solvent line of the column to be above the
added adsorbent. Unless indicated otherwise, chromatography was conducted
by flash column methods as described previously[55] utilizing a gradient of increasingly polar eluent specifically
indicated for each compound. Gradients were performed in a stepwise
fashion in 200 mL increments. For EtOAc/hexanes systems, the column
was charged with the defined starting eluent percentage. After the
sample was applied, 200 mL of the initial eluent was used, followed
by 200 mL at 10% increments until the product eluted into the final
gradient. For MeOH/DCM systems, the column was charged with the defined
starting eluent percentage. After the sample was applied, 200 mL of
the initial eluent was used, followed by 200 mL at 2.5% increments
until the product eluted into the final gradient. Isocratic separations
are denoted on an individual basis. TLC was used to monitor product
elution during flash column chromatography. Appropriate fractions
were combined, and solvents were evaporated in vacuo (rotary evaporator
under water aspirator vacuum) and then further dried by a vacuum pump
(0.5 mm Hg) for 24 h unless described otherwise. Samples that were
heated in a vacuum desiccator were equipped to a vacuum pump (0.5
mm Hg) and dried for a specified time and temperature denoted for
the individual procedure. Solvent solutions dried with Na2SO4 were stored in a sealed flask and allowed to sit for
at least 12 h. Upon completion, the drying agent was removed by vacuum
filtration, and the solvents were evaporated in vacuo and then further
dried by a vacuum pump (0.5 mm Hg) for 24 h. Samples reduced by hydrogenation
utilized a PARR hydrogenator. The psi of the H2 is provided
in the individual experimental detail. Melting points were performed
in triplicate on an electrothermal digital melting point apparatus
and are uncorrected. Proton (1H) and carbon (13C) NMR experiments were recorded on either a 600 MHz Bruker Avance,
Inova 600 MHz, or an Inova 400 MHz instrument. Samples were referenced
to TMS when present, or the solvent residual peak for 1H and 13C, respectively: (CDCl3; 7.27, 77.13;
DMSO-d6; 2.50, 39.51; MeOH-d4; 3.31, 49.15). 1HNMR chemical shifts were
given in ppm, and coupling constants (J values) were
expressed in hertz (Hz) using the following designations: s (singlet),
d (doublet), t (triplet), q (quartet), quin (quintet), sex (sextet),
sep (septet), dd (doublet of doublets), m (multiplet). The 13C chemical shifts are reported for each compound in the Experimental Section and in all cases confirm structure.
In a few cases 13C shifts were found to double-up in their
peak locations. Fluorine (19F) NMR was recorded on an Inova
400 MHz instrument at 376 MHz. Samples were referenced externally
to CFCl3. Purity for tested compounds (2a–2f, 2h, 2j–2q) was determined by combustion analysis (Atlantic Microlabs, Norcross
GA), or in the cases of 2g and 2i by HPLC.
All tested compounds possess ≥95% purity. Intermediate compounds
were determined to possess ≥95% purity by combustion analysis
except 4a and 6a. For these intermediates,
the mp and spectral data matched literature values. Observed values
for combustion analysis were considered acceptable within ±0.4%
of calculated values. Synthetic derivatives reported as solvates are
denoted in the text and were calculated by incorporating the minimal
amount of appropriate solvent that was subjected to the compound during
the purification process in order to be within the acceptable range
(±0.4%). HPLC was performed on an Alliance instrument (#2659)
equipped with a quaternary pump, an inline membrane degasser, autosampler,
and photodiode array (PDA) detector (#2996) from Waters Corporation
(Milford, MA). The column was a Nova-PakC18 column, 4 μm particle
size (150 mm × 3.9 mm). Details for HPLC analysis are described
in the individual procedures for 2g and 2i. High resolution mass spectrometry (HRMS) was performed by the University
of Michigan’s Department of Chemistry as a technical service.
Compounds 1a–1c, 10,
and 18 were described previously.[16,17] Compound 7 was prepared according to the literature.[17,18] Compounds 3, 15, and 22 were
purchased commercially from Sigma-Aldrich.
Compound 2f (35 mg, 0.0927 mmol) was suspended
in MeOH
(5 mL) and n class="Chemical">TFA (1 mL) and heated to reflux for 24 h. The volatiles
were evaporated in vacuo, and the sample was redissolved in H2O (5 mL) and neutralized with 1 NNaOH (determined by pH paper).
Purification by column chromatography (2.5% to 10% MeOH/DCM) and subsequent
drying at 40 °C in a vacuum desiccator for 24 h yielded an orange
solid (15 mg, 58%): mp 263–264 °C. TLC R 0.52 (10% MeOH/DCM). 1HNMR (600 MHz, d4-MeOH) δ 8.75 (m,
2H), 8.26–8.23 (d, 1H, J = 15.06 Hz), 7.96
(m, 2H), 7.40 (d, 1H, J = 1.8 Hz), 7.37–7.35
(d, 1H, J = 15 Hz), 7.16–7.14 (d, 1H, J = 8.4 Hz), 6.74–6.72 (dd, 1H, J1 = 8.4 Hz, J2 = 1.8 Hz),
2.56 (s, 3H). 13CNMR (150 MHz, MeOH-d4) δ 190.8, 151.2, 148.4, 147.6, 143.66, 143.21,
132.6, 128.7, 123.4, 114.4, 113.2, 111.6, 108.1, 12.1. HRMS ESI+ calculated m/z for C17H16N3O (M + H)+ 278.1288.
Found (M + H)+ 278.1286. HPLC analysis: retention time
=2.642 min; peak area, 99.48%; eluent A, 10 mM TEA solution with 0.1%
formic acid (pH 3); eluent B, CH3CN; isocratic (15% eluent
B) over 10 min with a flow rate of 1 mL min–1 and
detection at 280 nm; injection of 10 μL of 59 μM 2g.
Compound 2k (52 mg, 0.18 mmol)
was
dissolved in methanol (3 mL) and n class="Chemical">NaOH (1 N, 2 mL). The reaction mixture
was heated to reflux for 30 h. Upon completion, the final volume was
reduced by approximately one-half of the original volume in vacuo,
and the sample was stored at 4 °C overnight resulting in the
formation of an orange precipitate. The orange precipitate was collected,
washed dropwise with a 50% mixture of MeOH/H2O (10 mL,
chilled at 4 °C for 1 h), and dried at 40 °C in a vacuum
desiccator for 36 h to yield an orange salt (16 mg, 28%): mp >300
°C. TLC R 0.55
DCM/MeOH: Acetic acid (90:9:1). 1HNMR (600 MHz, DMSO-d6) δ 11.72 (s, 1H), 9.34–9.32 (d,
1H, J = 16.02 Hz), 8.79–8.78 (m, 2H), 7.88–7.87
(m, 2H), 7.43 (d, 1H, J = 1.8 Hz), 7.40–7.37
(m, 2H), 6.88–6.87 (dd, 1H, J1 =
8.76 Hz, J2 = 2.16 Hz), 3.86 (s, 3H); 13CNMR (150 MHz, DMSO-d6) δ
189.2, 163.5, 155.0, 150.5, 145.6, 144.8, 130.2, 126.5, 121.6, 114.8,
113.6, 112.4, 110.8, 103.8, 55.5. Elemental analysis calculated for
C18H13N2NaO4·2 H2O: C, 56.84; H, 4.51; N, 7.37. Found: C, 56.64; H,
4.60; N, 7.27. HRMS ESI– calculated m/z for C18H13N2O4 (M – H)− 321.0881. Found (M
– H)− 321.0884.
5-Methoxy-2-hydroxymethylindole-3-carboxaldehyde 27 (75 mg, 0.37 mmol), n class="Chemical">4-acetylpyridine (118 mg, 0.37 mmol),
and piperidine (82.6 mg, 0.37 mmol) were dissolved in methanol (1
mL) and heated to reflux. The reaction mixture was stirred for 45
min during which the solution became bright orange. The flask was
then removed from heat and allowed to stir for 1 h. A precipitate
formed which was collected, washed with ice-cold MeOH (5 mL), and
dried in a vacuum desiccator for 24 h to yield an orange solid (28
mg, 24%). mp 229–230 °C. TLC R 0.65 (6% MeOH/DCM), 0.21 (80% EtOAc/hexanes). 1HNMR (600 MHz, DMSO-d6) δ
11.96 (s, 1H), 8.82–8.81 (m, 2H), 8.17–8.14 (d, 1H, J = 15.36 Hz), 7.94–7.93 (m, 2H), 7.45–7.44
(d, 1H, J = 2.28 Hz), 7.38–7.34 (m, 2H), 6.88–6.86
(dd, 1H, J1 = 8.7 Hz, J2 = 2.34 Hz), 5.70–5.68 (t, 1H, J = 5.46 Hz), 4.85–4.84 (d, 2H, J = 5.46 Hz),
3.86 (s, 3H). 13CNMR (150 MHz, DMSO-d6) δ 188.2, 155.2, 150.6, 148.0, 145.0, 139.3, 131.0,
126.7, 121.4, 113.8, 113.0, 111.6, 108.3, 103.4, 55.57, 55.32. Elemental
Analysis calculated for C18H16N2O3·0.033 MeOH: C, 70.00; H, 5.26; N, 9.05. Found: C, 69.61;
H, 5.09; N, 8.90. This compound showed significant degradation when
stored in DMSO-d6 for 48 h as determined
by 1HNMR. After 48 h, ∼50% of 2p was
degraded, and the spectrum showed a number of unidentifiable compounds.
All other derivatives (1a–c, 2a–o, 2q) were stable in
prolonged exposure (up to 12 months) to DMSO. When stored in its isolated
precipitate form, this compound is stable.
2-Hydroxypropyl-5-methoxyindole-3-carboxaldehyde-O-acetate 32 (87 mg, 0.31 mmol) in n class="Chemical">MeOH (6 mL), piperidine (40 mg, 0.47
mmol), and 4-acetylpyridine (57 mg, 0.47 mmol) were heated to reflux
and stirred for 20 h. The solvent was reduced by approximately one-half
of the original volume in vacuo, and the flask was placed in a −20
°C freezer for 30 min. The resulting bright orange-red precipitate
was collected, washed with ice-cold MeOH (5 mL) and dried in a vacuum
desiccator for 24 h (16 mg, 15%): mp 221–223 °C. TLC R 0.23 (4% MeOH/DCM). 1HNMR (600 MHz, DMSO-d6) δ
11.87 (s, 1H), 8.81–8.80 (m, 2H), 8.12–8.10 (d, 1H, J = 15.24 Hz), 7.94–7.93 (m, 2H), 7.44 (d, 1H, J = 2.28 Hz), 7.40–7.37 (d, 1H, J = 15.24 Hz), 7.33–7.31 (d, 1H, J = 8.64
Hz), 6.87–6.85 (dd, 1H, J1 = 8.7
Hz, J2 = 2.4 Hz), 4.68–4.66 (t,
1H, J = 4.62 Hz), 3.86 (s, 3H), 3.48–3.45
(q, 2H, J = 6.0 Hz), 2.99–2.96 (t, 2H, J = 7.62 Hz), 1.86–1.81 (quin, 2H, J = 6.24 Hz). 13CNMR (150 MHz, DMSO-d6) δ 188.1, 155.2, 150.62, 149.80, 145.1, 139.5,
131.2, 126.4, 121.4, 113.01, 112.42, 111.1, 108.9, 103.7, 59.9, 55.6,
32.7, 22.7. Elemental Analysis for C20H20N2O3: C, 71.41; H, 5.99; N, 8.33. Found: C, 71.12;
H, 6.05; N, 8.34.
4-Ethoxy-2-methyl-1-nitrobenzene (4a)
3-Methyl-4-nitrophenol 3 (500 mg, 3.0 mmol)
was dissolved
inn class="Chemical">2-butanone (10 mL). Potassium carbonate (903 mg, 6.5 mmol) was
added to the solution followed by diethylsulfate (0.45 mL, 3.2 mmol).
The reaction mixture was heated at 70 °C for 2 h. Upon completion,
the mixture was cooled to rt, and aqueous ammonia (30%, 1 mL) was
added and stirred for 18 h at rt. The reaction mixture was filtered
and washed with 2-butanone (30 mL). The filtrate was collected and
evaporated in vacuo to yield a white solid (580 mg, 97%). mp 50–52 °C
(lit.[56] mp 51 °C). TLC R 0.65 (20% EtOAc/hexanes). 1HNMR (600 MHz, CDCl3) δ 8.11–8.09 (d, 1H, J = 8.88 Hz), 6.82–6.78 (m, 2H), 4.14–4.10
(q, 2H, J = 6.96 Hz), 2.65 (s, 3H), 1.48–1.46
(t, 3H, J = 6.95 Hz). 13CNMR (150 MHz,
CDCl3) δ 162.5, 142.0, 137.1, 127.6, 117.9, 112.2,
64.2, 21.8, 14.6.
2-Methyl-4-propoxy-1-nitrobenzene (4b)
Compound 3 (1.5 g, 9.8 mmol) was
dissolved in 2-butanone
(40 mL). n class="Chemical">Potassium carbonate (3.4 g, 24.5 mmol) was added, and the
solution was heated at reflux. After 5 min, 1-bromopropane (18.5 mmol,
2 mL) was added dropwise, and the reaction mixture continued to stir
for 18 h. The solvent was evaporated in vacuo, and the crude residue
was partitioned between EtOAc (150 mL) and saturated NaHCO3 (150 mL). The organic layer was separated, washed with brine (100
mL), and dried over Na2SO4. The resulting oil
was purified by column chromatography (0% to 20% EtOAc/hexanes) to
yield a yellow oil (1.89 g, 99%): TLC R 0.70 (20% EtOAc/hexanes). 1HNMR (600
MHz, CDCl3) δ 8.09–8.07 (d, 1H, J = 8.76 Hz), 6.79–6.77 (m, 2H), 3.99–3.97 (t, 2H, J = 6.54 Hz), 2.63 (s, 3H), 1.85–1.82 (sex, 2H, J = 7.44 Hz), 1.06–1.04 (t, 3H, J = 7.44 Hz). 13CNMR (150 MHz, CDCl3) δ
162.9, 142.2, 137.3, 127.8, 118.1, 112.4, 70.3, 22.60, 21.98, 10.6.
Elemental analysis calculated for C10H13NO3·0.03 hexanes: C, 61.81; H, 6.84; N, 7.08. Found: C,
62.20; H, 6.78; N, 7.23.
4-Isopropoxy-2-methyl-1-nitrobenzene
(4c)
This compound was prepared in a similn class="Chemical">ar
manner to that for 4b except that 2-bromopropane was
deployed for the alkyl ether
adduct and chromatography utilized a 0–15% gradient for elution
to yield a yellow oil (1.87 g, 98%): TLC R 0.68 (20% EtOAc/hexanes). 1HNMR (600
MHz, CDCl3) δ 8.08–8.07 (d, 1H, J = 8.82 Hz), 6.77–6.75 (m, 2H), 4.66–4.62 (sep, 1H, J = 6.06 Hz), 2.62 (s, 3H), 1.38–1.37 (d, 6H, J = 6.12 Hz). 13CNMR (150 MHz, CDCl3) δ 162.6, 141.7, 137.1, 127.6, 118.9, 112.8, 70.6, 21.84,
21.78. Elemental analysis calculated for C10H13NO3·0.06 hexanes: C, 62.10; H, 6.96; N, 6.99. Found:
C, 62.45; H, 6.69; N, 7.09.
4-Butoxy-2-methyl-1-nitrobenzene
(4d)
This compound was prepared in a similn class="Chemical">ar
manner to that for 4b except 1-bromobutane was deployed
for the alkyl ether adduct
and the partition step during workup utilized DCM (150 mL) and water
(150 mL) to provide a yellow oil (2.0 g, 99%): TLC R 0.70 (20% EtOAc/hexanes). 1HNMR (600 MHz, CDCl3) δ 8.09–8.07 (d, 1H, J = 8.88 Hz), 6.79–6.76 (m, 2H), 4.03–4.01
(t, 2H, J = 6.48 Hz), 2.63 (s, 3H), 1.80–1.78
(quin, 2H, J = 7.44 Hz), 1.53–1.47 (sex, 2H, J = 7.44 Hz), 1.0–0.97 (t, 3H, J = 7.38 Hz). 13CNMR (150 MHz, CDCl3) δ
162.7, 141.9, 137.1, 127.6, 117.9, 112.2, 68.3, 31.0, 21.8, 19.1,
13.8. Elemental analysis calculated for C11H15NO3: C, 63.14; H, 7.23; N, 6.69. Found: C, 63.25; H, 7.09;
N, 6.69.
4-Ethoxy-2-methylaniline (5a)
Compound 4a (250 mg, 1.38 mmol) was dissolved
in EtOAc (10 mL) and
n class="Chemical">MeOH (10 mL) and transferred to a 250 mL hydrogenation flask. 10%
Pd/C (25 mg, 10% w/w) was added, and the sample was hydrogenated for
4 h at 35 psi H2. Upon completion, the mixture was filtered
over a bed of Celite and then concentrated in vacuo. The residue was
dissolved in EtOAc (50 mL) and washed with NaHCO3 (50 mL
× 3). The organic layer was dried over Na2SO4 to yield a dark-red oil (192 mg, 92%): TLC R 0.30 (20% EtOAc/hexanes). 1NMR
(600 MHz, CDCl3) δ 6.67 (s, 1H), 6.62 (m, 2H), 3.97–3.93
(q, 2H, J = 6.96 Hz), 3.42 (s, 2H), 2.16 (s, 3H),
1.38–1.35 (t, 3H, J = 7.02 Hz). 13CNMR (150 MHz, CDCl3) δ 152.3, 138.1, 124.4, 117.45,
116.34, 113.1, 64.2, 17.9, 15.2. Elemental analysis calculated for
C9H13NO: C, 71.49; H, 8.67; N, 9.26. Found:
C, 71.42; H, 8.76; N, 9.04.
2-Methyl-4-propoxyaniline
(5b)
Compound 4b (3.78 g, 19.4
mmol) was dissolved in n class="Chemical">EtOAc (20 mL) and
MeOH (20 mL) and transferred to a 250 mL hydrogenation flask. 10%
Pd/C (378 mg, 10% w/w) was added, and the sample was hydrogenated
for 4 h at 35 psi H2. Upon completion, the mixture was
filtered over a bed of Celite and then concentrated in vacuo. The
residue was dissolved in EtOAc (100 mL) and washed with NaHCO3 (100 mL × 3). The organic layer was separated and then
dried over Na2SO4 to provide a crude brown oil
which was further purified by chromatography (20% to 50% EtOAc/hexanes)
to yield an amber oil (2.72 g, 85%): TLC R 0.27 (20% EtOAc/hexanes). 1NMR (600 MHz,
CDCl3) δ 6.67 (s, 1H), 6.62 (m, 2H), 3.85–3.38
(t, 2H, J = 6.66 Hz), 2.16 (s, 3H), 1.79–1.73
(sex, 2H, J = 7.44 Hz), 1.02–1.00 (t, 3H, J = 7.44 Hz). 13CNMR (150 MHz, CDCl3) δ 152.3, 137.8, 124.1, 117.2, 116.1, 112.9, 70.1, 22.7, 17.7,
10.5. Elemental analysis calculated for C10H15NO: C, 72.69; H, 9.15; N, 8.48. Found: C, 72.42; H, 9.14; N, 8.46.
4-Isopropoxy-2-methylaniline (5c)
This
compound was prepared from n class="Chemical">4c (3.64 g, 18.7 mmol) in
a manner similar to that for 5b to yield an amber oil
(2.5 g, 81%): TLC R 0.33
(20% EtOAc/hexanes). 1NMR (600 MHz, CDCl3) δ
6.67 (s, 1H), 6.62 (m, 2H), 4.39–4.35 (sep, 1H, J = 6.06 Hz), 2.15 (s, 3H), 1.29–1.28 (d, 6H, J = 6.06 Hz). 13CNMR (150 MHz, CDCl3) δ
150.8, 138.0, 124.1, 119.4, 116.10, 115.09, 71.0, 22.2, 17.7. Elemental
analysis calculated for C10H15NO: C, 72.69;
H, 9.15; N, 8.48. Found: C, 72.75; H, 9.11; N, 8.33.
4-Butoxy-2-methylaniline
(5d)
This compound
was prepared from 4d (1.92 g, 9.2 mmol) in a manner similn class="Chemical">ar
to that for 5b yielding an amber oil (1.48 g, 90%): TLC R 0.28 (20% EtOAc/hexanes). 1NMR (600 MHz, CDCl3) δ 6.67 (s, 1H), 6.62
(m, 2H), 3.89–3.87 (t, 2H, J = 6.54 Hz), 2.16
(s, 3H), 1.73–1.70 (quin, 2H, J = 6.6 Hz),
1.49–1.45 (sex, 2H, J = 7.5 Hz), 0.97–0.95
(t, 3H, J = 7.38 Hz). 13CNMR (150 MHz,
CDCl3) δ 152.4, 137.8, 124.2, 117.24, 116.13, 112.9,
68.3, 31.5, 19.3, 17.7, 13.9. Elemental analysis calculated for C11H17NO: C, 73.70; H, 9.56; N, 7.81. Found: C, 73.55;
H, 9.51; N, 7.96.
Compound 5a (210 mg, 1.39
mmol) and n class="Chemical">di-tert-butyl-dicarbonate (334 mg, 1.53
mmol) in THF (10 mL) were heated to reflux for 20 h. The reaction
mixture was concentrated in vacuo and redissolved in DCM (30 mL).
This mixture was washed with saturated NaHCO3 (30 mL) and
brine (30 mL). The organic layer was separated and dried over Na2SO4 to produce a dark-red oil which was purified
by chromatography (0% to 20% EtOAc/hexanes) to yield an orange solid
(293 mg, 84%): mp 66–68 °C (lit.[56] mp 66 °C). TLC R 0.50 (20% EtOAc/hexanes). 1HNMR (600 MHz, CDCl3) δ 7.51 (s, 1H), 6.72 (m, 2H), 6.06 (s, 1H), 4.01–3.97
(q, 2H, J = 6.96 Hz), 2.22 (s, 3H), 1.53 (s, 9H),
1.40–1.37 (t, 3H, J = 6.9 Hz). 13CNMR (150 MHz, CDCl3) δ 155.8, 153.7, 146.7, 129.0,
124.0, 116.6, 112.2, 80.1, 63.6, 28.4, 18.1, 14.9.
This compound was prepared from n class="Chemical">5c in a similar manner to that for 6b except that chromatography
used a gradient of 0% to 15%. A white solid was obtained (3.78 g,
99%): mp 57–59 °C. TLC R 0.57
(20% EtOAc/hexanes). 1HNMR (600 MHz, CDCl3)
δ 7.50 (s, 1H), 6.72–6.70 (m, 2H), 6.06 (s, 1H), 4.50–4.45
(sep, 1H, J = 6.06 Hz), 2.21 (s, 3H), 1.59 (s, 9H),
1.31–1.30 (d, 6H, J = 6.06 Hz). 13CNMR (150 MHz, CDCl3) δ 154.63, 153.72, 131.2,
129.0, 123.9, 118.2, 113.7, 80.1, 70.1, 28.4, 22.1, 18.0. Elemental
analysis calculated for C15H23NO3: C, 67.90; H, 8.74; N, 5.28. Found: C, 67.97; H, 8.76; N, 5.24.
Compound 5d (1.18 g, 6.6 mmol)
and di-tert-butyl dicarbonate (1.58 g, 7.24 mmol)
inn class="Chemical">THF (50 mL) were heated to reflux for 24 h. The solvent was evaporated
in vacuo, and the residue was dissolved in EtOAc (50 mL) and washed
with brine (50 mL × 3). The organic layer was separated and then
dried over Na2SO4. The residue was purified
by chromatography (0–20% EtOAc/hexanes) to provide a light-orange
solid (4.06 g, 98%): mp 53–55 °C. TLC R 0.65 (20% EtOAc/hexanes). 1HNMR (600 MHz, CDCl3) δ 7.50 (s, 1H), 6.72–6.70
(m, 2H), 6.06 (s, 1H), 3.93–3.91 (t, 2H, J = 6.54 Hz), 2.22 (s, 3H), 1.76–1.71 (quin, 2H, J = 6.6 Hz), 1.50 (s, 9H), 1.49–1.44 (m, 2H), 0.98–0.95
(t, 3H, J = 7.38 Hz). 13CNMR (150 MHz,
CDCl3) δ 156.0, 153.7, 131.1, 128.9, 124.0, 116.5,
112.2, 80.1, 67.8, 31.3, 28.4, 19.2, 18.0, 13.8. Elemental analysis
calculated for C16H25NO3: C, 68.79;
H, 9.02; N, 5.01. Found: C, 68.87; H, 8.94; N, 5.02.
5-Ethoxy-2-methylindole
(8a)
Compound 6a (900 mg, 3.58
mmol) was dissolved in THF (15 mL) under
an atmosphere of n class="Chemical">Argon. The solution was cooled to −40 °C
over 10 min and sec-butyllithium (1.4 M, 5.37 mL)
was added slowly as to maintain an internal temperature of < −25
°C. After reaching 1 equiv of sec-butyllithium
(2.7 mL), the reaction mixture turned a bright yellow signifying deprotonation
of the amidenitrogen. The reaction mixture was then cooled to −50
°C, and a solution of N-methoxy-N-methylacetylamide 7 (442.8 mg, 4.78 mmol) in THF (3
mL) was added over 5 min. The reaction mixture was warmed to −10
°C over 30 min. The mixture was partitioned between Et2O (75 mL) and 0.5 NHCl (75 mL). The aqueous layer was separated
and extracted an additional two times with Et2O (50 mL).
The Et2O phases were combined and washed with brine (75
mL) and then dried over Na2SO4 to yield a dark-brown
oil. The crude intermediate was dissolved in DCM (20 mL). TFA (3 mL)
was added to the mixture which was then stirred at rt for 48 h. Upon
completion, the reaction mixture was added to a separatory funnel
and washed with NaHCO3 (50 mL) followed by brine (50 mL).
The organic layer was separated and then dried over Na2SO4 to provide a crude black oil (1 g) which was purified
by chromatography (0–20% EtOAc/hexanes) to yield a brown solid
(335 mg, 53%): mp 88–90 °C. TLC R 0.48 (20% EtOAc/hexanes). 1HNMR (600 MHz, CDCl3) δ 7.72 (s, 1H), 7.16–7.14
(d, 1H, J = 8.64 Hz), 6.99 (d, 1H, J = 2.4 Hz), 6.77–6.75 (dd, 1H, J1 = 8.7 Hz, J2 = 2.4 Hz), 6.13 (s, 1H),
4.07–4.04 (q, 2H, J = 6.96 Hz), 2.41 (s, 3H),
1.43–1.40 (t, 3H, J = 6.96 Hz). 13CNMR (150 MHz, CDCl3) δ 153.3, 135.8, 131.2, 129.5,
111.33, 110.75, 103.1, 100.3, 64.2, 15.1, 13.8. Elemental analysis
calculated for C11H13NO: C, 75.40; H, 7.48;
N, 7.99. Found: C, 75.11; H, 7.64; N, 7.46.
2-Methyl-5-propoxyindole
(8b)
This compound
was prepared from 6b (1.00 g, 3.77 mmol) in a manner
similn class="Chemical">ar to that for 8a except: after the addition of
TFA the mixture was stirred for 24 h at rt then heated at reflux for
an additional 2 h to ensure complete BOC indole deprotection, and
the gradient for chromatography was 0–15%. A yellow solid was
obtained (0.28 g, 39%): mp 66–67 °C. TLC R 0.50 (20% EtOAc/hexanes). 1HNMR (600
MHz, CDCl3) δ 7.72 (s, 1H), 7.16–7.14 (d,
1H, J = 8.7 Hz), 6.99 (d, 1H, J =
2.34 Hz), 6.77–6.76 (dd, 1H, J1 = 8.7 Hz, J2 = 2.4 Hz), 6.13 (s, 1H),
3.96–3.93 (t, 2H, J = 6.66 Hz), 2.41 (s, 3H),
1.84–1.78 (sex, 2H, J = 7.32 Hz), 1.06–1.03
(t, 3H, J = 7.44 Hz). 13CNMR (150 MHz,
CDCl3) δ 153.5, 135.8, 131.1, 129.5, 111.3, 110.7,
103.1, 100.3, 70.4, 22.8, 13.8, 10.6. Elemental analysis calculated
for C12H15NO: C, 76.16; H, 7.99; N, 7.40. Found:
C, 76.13; H, 7.90; N, 7.36.
5-Isopropoxy-2-methylindole
(8c)
This
compound was prepared from n class="Chemical">6c (1.00 g, 3.77 mmol) in
a manner similar to that for 8b to yield a yellow solid
(0.43 g, 60%): mp 64–65 °C. TLC R 0.52 (20% EtOAc/hexanes). 1HNMR (600 MHz, CDCl3) δ 7.69 (s, 1H), 7.14–7.13
(d, 1H, J = 8.7 Hz), 7.01 (d, 1H, J = 2.28 Hz), 6.75–6.74 (dd, 1H, J1 = 8.64 Hz, J2 = 2.34 Hz), 6.12 (s, 1H),
4.50–4.46 (sep, 1H, J = 6.06 Hz), 2.40 (s,
3H), 1.33–1.32 (d, 6H, J = 6.06 Hz). 13CNMR (150 MHz, CDCl3) δ 152.2, 135.8, 131.6,
129.7, 113.1, 110.6, 106.2, 100.3, 71.5, 22.3, 13.8. Elemental analysis
calculated for C12H15NO: C, 76.16; H, 7.99;
N, 7.40. Found: C, 76.12; H, 7.86; N, 7.29.
5-Butoxy-2-methylindole
(8d)
This compound
was prepared from 6d in a manner similn class="Chemical">ar to that for 8a to yield a brown solid (0.356 g, 49%): mp 58–60
°C. TLC R 0.50
(20% EtOAc/hexanes). 1HNMR (600 MHz, CDCl3)
δ 7.72 (s, 1H), 7.16–7.14 (d, 1H, J =
8.7 Hz), 6.99 (d, 1H, J = 2.4 Hz), 6.77–6.75
(dd, 1H, J1 = 8.7 Hz, J2 = 2.4 Hz), 6.13 (s, 1H), 4.00–3.98 (t, 2H, J = 6.6 Hz), 2.41 (s, 3H), 1.80–1.75 (quin, 2H, J = 6.6 Hz), 1.54–1.49 (sex, 2H, J = 7.5 Hz), 0.99–0.96 (t, 3H, J = 7.44 Hz). 13CNMR (150 MHz, CDCl3) δ 153.6, 135.8, 131.1,
129.5, 111.3, 110.7, 103.0, 100.3, 68.5, 31.6, 19.3, 13.94, 13.81.
Elemental analysis calculated for C13H17NO:
C, 76.81; H, 8.43; N, 6.89. Found: C, 76.84; H, 8.31; N, 6.78.
5-Ethoxy-2-methylindole-3-carboxaldehyde
(9a)
DMF (2 mL) was cooled to 0 °C. n class="Chemical">POCl3 (0.5 mL) was
added and the reaction mixture was stirred for 10 min. A solution
of compound 8a (250 mg, 1.43 mmol) in DMF (3 mL) was
added dropwise over 10 min and then stirred for an additional 40 min
while warming to rt. The reaction mixture was added to ice-cold 1
NNaOH (35 mL), and the solution was stirred for 30 min in an ice-bath.
The resulting precipitate was collected, washed with ice-cold H2O (20 mL), and dried at 40 °C for 24 h in a vacuum desiccator
to yield a light-brown solid (238 mg, 82%): mp 199–200 °C.
TLC R 0.51 (80% EtOAc/hexanes). 1HNMR (600 MHz, DMSO-d6) δ
11.85 (s, H) 10.00 (s, 1H), 7.55 (d, 1H, J = 2.34
Hz), 7.27–7.25 (d, 1H, J = 8.7 Hz), 6.78–6.76
(dd, 1H, J1 = 8.7 Hz J2 = 2.4 Hz), 4.03–3.99 (q, 2H, J = 6.96 Hz), 2.64 (s, 3H), 1.35–1.33 (t, 3H, J = 6.96 Hz). 13CNMR (150 MHz, DMSO-d6) δ 184.5, 155.2, 149.0, 130.5, 126.8, 114.1, 112.71,
112.52, 103.6, 63.7, 15.3, 12.0. Elemental analysis calculated for
C12H13NO2: C, 70.92; H, 6.45; N,
6.89. Found: C, 70.66; H, 6.57; N, 6.97.
2-Methyl-5-propoxyindole-3-carboxaldehyde
(9b)
This compound was prepared from n class="Chemical">8b (263 mg, 1.39 mmol)
in a manner similar to that for 9a except for after the
dropwise addition of substitutedindole, the solution was stirred
for 1 h. A cream-colored powder was obtained (228 mg, 75%): mp 156–158
°C. TLC R 0.52
(75% EtOAc/hexanes). 1HNMR (600 MHz, DMSO-d6) δ 11.85 (s, 1H), 10.00 (s, 1H), 7.55 (d, 1H, J = 2.46 Hz), 7.27–7.25 (d, 1H, J = 8.7 Hz), 6.79–6.77 (dd, 1H, J1 = 8.7 Hz, J2 = 2.52 Hz), 3.92–3.90
(t, 2H, J = 6.54 Hz), 2.64 (s, 3H), 1.77–1.71
(sex, 2H, J = 7.38 Hz), 1.00–0.98 (t, 3H, J = 7.38 Hz). 13CNMR (150 MHz, DMSO-d6) δ 184.0, 154.9, 148.5, 130.0, 126.4,
113.6, 112.26, 112.04, 103.2, 69.3, 22.2, 11.5, 10.5. Elemental analysis
calculated for C13H15NO2: C, 71.87;
H, 6.96; N, 6.45. Found: C, 71.74; H, 6.81; N, 6.42.
5-Isopropoxy-2-methylindole-3-carboxaldehyde
(9c)
This compound was prepared from n class="Chemical">8c (347 mg,
1.83 mmol) in a manner similar to that for 9a except
for after the dropwise addition of substitutedindole, the solution
was stirred for 90 min. A cream-colored powder was obtained (313 mg,
78%): mp 179–181 °C. TLC R 0.43 (75% EtOAc/hexanes). 1HNMR (600
MHz, DMSO-d6) δ 11.84 (s, 1H), 10.00
(s, 1H), 7.56 (d, 1H, J = 2.4 Hz), 7.26–7.25
(d, 1H, J = 8.64 Hz), 6.77–6.76 (dd, 1H, J1 = 8.7 Hz, J2 =
2.46 Hz), 4.56–4.50 (sep, 1H, J = 6 Hz), 2.64
(s, 3H), 1.27–1.26 (d, 6H, J = 6 Hz). 13CNMR (150 MHz, DMSO-d6) δ
184.0, 153.4, 148.6, 130.1, 126.4, 113.58, 113.56, 112.0, 105.5, 69.9,
22.0, 11.5. Elemental analysis calculated for C13H15NO2: C, 71.87; H, 6.96; N, 6.45. Found: C, 71.70;
H, 6.83; N, 6.60.
5-Butoxy-2-methylindole-3-carboxaldehyde
(9d)
This compound was prepared from 8d (300 mg, 1.48 mmol)
in a manner similn class="Chemical">ar to that for 9b to yield a cream-colored
powder (320 mg, 94%): mp 160–161 °C. TLC R 0.45 (3:1 EtOAc/hexanes). 1HNMR (600 MHz, DMSO-d6) δ 11.85
(s, 1H), 10.00 (s, 1H), 7.55 (d, 1H, J = 2.46 Hz),
7.26–7.25 (d, 1H, J = 8.7 Hz), 6.79–6.77
(dd, 1H, J1 = 8.7 Hz, J2 = 2.46 Hz), 3.96–3.94 (t, 2H, J = 6.48 Hz), 2.64 (s, 3H), 1.73–1.68 (quin, 2H, J = 6.48 Hz), 1.49–1.42 (sex, 2H, J = 7.38
Hz), 0.95–0.93 (t, 3H, J = 7.38 Hz). 13CNMR (150 MHz, DMSO-d6) δ
185.0, 154.9, 148.5, 130.0, 126.4, 113.6, 112.26, 112.04, 103.2, 67.4,
31.0, 18.8, 13.8, 11.5. Elemental analysis calculated for C14H17NO2: C, 72.70; H, 7.41; N, 6.06. Found:
C, 72.87; H, 7.47; N, 6.13.
5-(N,N-Diacetyl-amino)-2-methylindole (11)
5-Amino-2-methylindole 10 (200 mg,
1.37 mmol) was dissolved inn class="Chemical">acetic anhydride (25 mL, 2.65 mmol), and
the solution was heated at 60 °C for 20 h. Upon completion, the
volatiles were evaporated and the oil partitioned between DCM (15
mL) and water (10 mL). The organic layer was separated, dried over
Na2SO4, and purified by chromatography (isocratic
50% EtOAc/hexanes) to yield an orange-brown solid (200 mg, 64%): mp
130–134 °C. TLC R 0.33 (1:1 EtOAc/hexanes). 1HNMR (400 mHz, CDCl3) δ 8.12 (s, 1H), 7.26 (s, 1H), 7.25–7.24 (d,
1H, J = 0.8 Hz), 6.82–6.80 (dd, 1H, J1 = 8.4 Hz, J2 =
2 Hz), 6.21 (s, 1H), 2.42 (s, 3H), 2.31 (s, 6H); 13CNMR
(150 MHz, CDCl3) δ 174.2, 137.3, 135.8, 130.9, 129.6,
120.3, 119.0, 111.6, 100.2, 27.1, 13.5. Elemental analysis calculated
for C13H14N2O2: C, 67.81;
H, 6.13; N, 12.17. Found: C, 67.73; H, 6.16; N 11.99.
5-(N-Boc-Amino)-2-methylindole (12)
5-Amino-2-methylindole 10 (400 mg, 1.62 mmol)
and n class="Chemical">di-tert-butyl-dicarbonate (712 mg, 3.26 mmol)
were dissolved in CH3CN (12 mL) and stirred at rt for 20
h. The volatiles were evaporated in vacuo, the residue was purified
by chromatography (isocratic 50% EtOAc/hexanes), and the product was
dried in a vacuum desiccator for 20 h to yield a light-brown solid
(615 mg, 91%): mp 134–137 °C. TLC R 0.66 (1:1 EtOAc/hexanes). 1HNMR (600 MHz, CDCl3) δ 7.85 (s, 1H), 7.54 (s, 1H),
7.14–7.13 (d, 1H, J = 8.4 Hz), 7.01–6.99
(d, 1H, J = 7.8 Hz), 6.44 (s, 1H), 6.13 (s, 1H),
2.39 (s, 3H), 1.53 (s, 9H); 13CNMR (150 MHz, CDCl3) δ 153.9, 136.4, 133.2, 130.5, 129.3, 114.5, 110.6,
100.2, 80.0, 28.6, 13.7. Elemental analysis calculated for C14H18N2O2: C, 68.27; H, 7.37; N, 11.37.
Found: C, 68.30; H, 7.39; N 11.21.
5-Acetamido-2-methylindole-3-carboxaldehyde
(13)
This compound was prepared from 11 (320 mg,
1.39 mmol) in a manner similn class="Chemical">ar to that for 9a except
for after dropwise addition of substitutedindole the solution was
stirred for 2 h to yield an orange precipitate. The sample was further
purified by chromatography (0% to 10% MeOH/DCM) to yield a white solid
(222 mg, 74%): Mp 263 °C (darkening). TLC R 0.46 (10% MeOH/DCM). 1HNMR (600
MHz, CD3OD) δ 9.99 (s, 1H), 8.18 (d, 1H, J = 1.8 Hz), 7.47–7.45 (dd, 1H, J1 = 8.4 Hz J2 = 1.8 Hz), 7.30–7.29
(d, 1H, J = 9 Hz), 2.70 (s, 3H), 2.14 (s, 3H). 13CNMR (150 MHz, CD3OD) δ 186.2, 171.6, 151.4,
135.03, 134.29, 127.4, 118.6, 115.6, 113.8, 112.3, 23.7, 11.8. Elemental
analysis calculated for C12H12N2O2·0.15 H2O: C, 65.83; H, 5.66; N, 12.80. Found:
C, 65.75; H, 5.59; N, 12.42.
This compound was prepared from 12 (615 mg, 2.24 mmol) in a manner similn class="Chemical">ar to that for 9a except for after the dropwise addition of substitutedindole the
solution was stirred for 4 h to provide an orange precipitate. The
precipitate was further purified by chromatography (isocratic 5% MeOH/DCM)
to yield a white solid (250 mg, 36%): mp 201–203 °C. TLC R 0.33 (75% EtOAc/hexanes). 1HNMR (600 MHz, DMSO-d6) δ
9.70 (s, 1H), 8.08 (s, 1H), 7.26–7.24 (m, 2H), 2.69 (s, 3H),
1.52 (s, 9H). 13CNMR (150 MHz, DMSO-d6) δ 184.4, 153.4, 149.4, 134.5, 131.6, 125.9, 115.6,
113.9, 111.4, 110.3, 79.0, 28.5, 11.7. Elemental analysis calculated
for C15H18N2O3·0.125
H2O: C, 65.14; H, 6.65; N, 10.13. Found: C, 64.82; H, 6.81;
N, 10.14. HRMS ESI+ calculated for C15H18N2O3 (M + H)+ 275.1390;
(M + Na)+ 297.1210. Found (M + H)+ 275.1393;
(M + Na)+ 297.1210.
N-Boc-5-(aminomethyl)indole
(16)
Compound 15 (200 mg, 1.37
mmol) and di-tert-butyl dicarbonate (358 mg, 1.64
mmol) inn class="Chemical">CH3CN (10 mL) were stirred at rt for 48 h. Upon
completion, solvents
were evaporated in vacuo. The residue was purified by chromatography
(10–50% EtOAc/hexanes) and dried at 40 °C in a vacuum
desiccator for 24 h to yield a colorless oil (297 mg, 88%): TLC R 0.69 (50% EtOAc/hexanes). 1HNMR (600 MHz, CDCl3) δ 8.51 (s, 1H), 7.53
(s, 1H), 7.32–7.31 (d, 1H, J = 8.4 Hz), 7.18
(s, 1H), 7.12–7.11 (d, 1H, J = 7.8 Hz), 6.50
(s, 1H), 4.89 (s, 1H), 4.41–4.40 (d, 2H, J = 4.2 Hz), 1.48 (s, 9H). 13CNMR (150 MHz, CDCl3) δ 156.2, 135.4, 130.2, 128.2, 125.0, 122.2, 120.0, 111.5,
102.6, 79.5, 45.5, 28.7. Elemental analysis calculated for C14H18N2O2: C, 68.27; H, 7.37; N, 11.37.
Found: C, 68.11; H, 7.24; N, 11.17. Although we obtained a colorless
oil, a previous report notes a light yellow solid with a mp 86–89
°C.[57]
N-Boc-5-(aminomethyl)indole-3-carboxaldehyde
(17)
This compound was prepared from 16 (297 mg, 1.21 mmol) in a manner similn class="Chemical">ar to that for 9a to yield a cream-colored solid (243 mg, 73%): mp 126–128
°C. TLC R 0.50
(80% EtOAc/hexanes). 1HNMR (600 MHz, DMSO-d6) δ 12.10 (s, 1H), 9.91 (s, 1H), 8.27 (m, 1H),
8.00 (s, 1H), 7.45–7.43 (m, 2H), 7.17–7.15 (d, 1H, J = 8.4 Hz), 4.22–4.20 (d, 2H, J = 6 Hz), 1.40 (s, 9H). 13CNMR (150 MHz, CDCl3) δ 184.7, 155.7, 138.6, 136.0, 134.0, 124.0, 123.1, 119.2,
118.0, 112.0, 77.6, 43.7, 28.2. Elemental analysis calculated for
C15H18N2O3: C, 65.68;
H, 6.61; N, 10.21. Found: C, 65.75; H, 6.61; N, 10.20.
N-Methoxy-N-methyltrifluoroacetamide
(19)
O,N-Dimn class="Chemical">ethylhydroxylamine
hydrochloride (0.77g, 7.8 mmol) and trifluoroacetic anhydride (1.5
g, 1.0 mL, 7.1 mmol) in anhydrous DCM (25 mL) were stirred for 15
min at 0 °C. Pyridine (1.27 mL, 15.7 mmol) was added dropwise
over 15 min. The reaction was then removed from the ice bath and stirred
for 3.5 h at rt. The reaction mixture was washed with 0.5 NHCl (50
mL × 2), saturated NaHCO3 (50 mL) and brine (50 mL).
The organic layer was separated and dried over Na2SO4. Bulb-to-bulb distillation was performed on the sample under
water aspirator vacuum to obtain a colorless oil (578 mg, 52%): 1HNMR (600 MHz, CDCl3) δ 3.78 (s, 3H), 3.30
(s, 3H). 13CNMR (150 MHz, CDCl3) δ 156.9
(q, 2JFC = 39 Hz), 116.1(q, 1JFC = 284 Hz), 62.2, 32.9. 19F NMR (376 MHz, CDCl3) δ −72.2 (s,
3F). Elemental analysis calculated for C4H6F3NO2: C, 30.58; H, 3.85; N, 8.92. Found: C, 30.65;
H, 4.04; N, 8.83.
5-Methoxy-2-trifluoromethylindole (20)
This compound was prepared from 18 (1.0 g, 4.2 mmol)
in a manner similn class="Chemical">ar to that for 8a except Weinreb amide 19 (726 mg, 4.6 mmol) was utilized as the acylating agent
to yield a yellow powder (0.390 g, 43%): mp 55 °C. TLC R 0.52 (20% EtOAc/hexanes). 1HNMR (600 MHz, CDCl3) δ 8.30 (s, 1H), 7.32–7.31
(d, 1H, J = 8.9 Hz), 7.10 (d, 1H, J = 2.4 Hz), 7.00–6.98 (dd, 1H, J1 = 8.9 Hz, J2 = 2.4 Hz), 6.85 (s, 1H),
3.85 (s, 3H). 13CNMR (150 MHz, CDCl3) δ
154.9, 131.2, 127.09, 126.15 (q, 2JFC = 38 Hz), 121.2 (q, 1JFC = 266 Hz), 115.8, 112.6, 104.0 (q, 3JFC = 3 Hz), 102.7, 55.8. 19F NMR (376 MHz,
CDCl3) δ −60.9 (s, 3F). Elemental analysis
calculated for C10H8F3NO· 0.04
hexanes: C, 56.26; H, 3.95; N, 6.41. Found: C, 56.63; H, 3.95; N,
6.37.
DMF (1 mL) was cooled to 0 °C and
n class="Chemical">POCl3 (0.25 mL) was added and stirred for 10 min. Compound 20 (336 mg, 1.69 mmol) in DMF (2 mL) was added dropwise over
10 min.
The solution was stirred for an additional 1 h warming to rt and then
heated to 85 °C and allowed to react for an additional 4 h. The
solution was poured into ice-cold 1 NNaOH (40 mL) and stirred for
30 min in an ice-bath resulting in the formation of a precipitate.
The precipitate was collected, washed with cold H2O, and
dried overnight at 40 °C in a vacuum desiccator to yield a cream-colored
solid. The sample was further purified by chromatography (7–20%
EtOAc/hexanes) to obtain a white solid (178 mg, 43%): mp 236–239
°C. TLC R 0.51
(30% EtOAc/hexanes). 1HNMR (600 MHz, MeOH-d4) δ 10.22 (s, 1H), 7.79 (d, 1H, J = 2.46 Hz), 7.45–7.43 (d, 1H, J = 9 Hz),
7.06–7.04 (dd, 1H, J1 = 8.94 Hz, J2 = 2.52 Hz), 3.86 (s, 3H). 13CNMR
(150 MHz, MeOH-d4) δ 186.2, 159.0,
132.0, 127.1, 123.4, 121.6, 118.2, 117.2, 114.8, 104.1, 56.2. 19F NMR (376 MHz, CDCl3) δ −58.7 (s,
3F). Elemental analysis calculated for C11H8F3NO2: C, 54.33; H, 3.32; N, 5.76. Found: C,
54.53; H, 3.32; N, 5.76.
Methyl 5-Methoxyindole-2-carboxylate
(23a)
Methanolic HCl was prepn class="Chemical">ared by bubbling
anhydrous HCl (g) into anhydrous
methanol (50 mL) for 5 min. 5-Methoxyindole-2-carboxylic acid 22 (2.0 g, 10.5 mmol) was added to the solution, and the reaction
was heated to reflux for 24 h. The mixture was concentrated in vacuo
and partitioned between 1 NNaOH (15 mL) /saturated NaHCO3 solution (85 mL) and EtOAc (100 mL). The organic layer was separated,
washed with brine (50 mL), and then dried over Na2SO4. The resulting crude brown solid was purified by chromatography
(20% to 75% EtOAc/hexane) to produce a light-brown solid (1.69 g,
78%). mp 183–186 °C (lit.[58] 177–178 °C). TLC R 0.67 in 50% EtOAc/Hex. 1HNMR (600 MHz, MeOH-d4) δ 7.33–7.31 (d, 1H, J = 9 Hz), 7.08 (m, 2H), 6.93–6.91 (dd, 1 H, J1 = 9 Hz, J2 = 2.5
Hz), 3.90 (s, 3H), 3.80 (s, 3 H). 13CNMR (150 MHz, MeOH-d4) δ 164.1, 156.1, 134.6, 129.07, 128.85,
117.9, 114.2, 109.1, 103.2, 56.1, 52.3. Elemental Analysis calculated
for C11H11NO3: C, 64.38; H, 5.40;
N, 6.83. Found: C, 64.55; H, 5.45; N; 6.95.
Ethyl
5-Methoxyindole-2-carboxylate (23b)
This compound
was prepared in a manner similn class="Chemical">ar to that for 23a except
ethanolic HCl was deployed as the reactive solvent
to produce a light-brown solid (1.74 g, 76%): mp 158–160 °C
(lit.[22] 156–159 °C). TLC R 0.45 (25% EtOAc/hexanes). 1HNMR (600 MHz, CDCl3) δ 8.88 (s, 1H), 7.32–7.31
(d, 1H, J = 8.9 Hz), 7.14 (m, 1H), 7.08 (d, 1H, J = 2.3 Hz), 7.01–6.99 (dd, 1 H, J1 = 8.9 Hz, J2 = 2.5 Hz),
4.42–4.39 (q, 2H, J = 7.1 Hz), 3.85 (s, 3
H), 1.42–1.40 (t, 3H, J = 7.1 Hz). 13CNMR (150 MHz, CDCl3) δ 161.9, 154.7, 132.1, 127.87,
127.84, 117.0, 112.8, 108.2, 102.5, 61.0, 55.7, 14.4. Elemental Analysis
calculated for C12H13NO3: C, 65.74;
H, 5.98; N, 6.39. Found: C, 66.02; H, 6.09; N; 6.45.
Propyl
5-Methoxyindole-2-carboxylate (23c)
To a suspension
of 22 (670 mg, 3.50 mmol) in anhydrous n-PrOH (20 mL) was added n class="Chemical">HCl (4 N in dioxane, 2.5 mL). The
reaction mixture was heated at reflux for 36 h. Upon completion, volatiles
were evaporated in vacuo, and the resulting oil was purified by chromatography
(0% to 20% EtOAc/hexanes) to produce a white powder (721 mg, 88%):
mp 104–107 °C. TLC R 0.51
(20% EtOAc/hexanes). 1HNMR (600 MHz, CDCl3)
δ 8.82 (s, 1H), 7.32–7.31 (d, 1H, J =
8.94 Hz), 7.15 (m, 1H), 7.08 (d, 1H, J = 2.4 Hz),
7.01–6.99 (dd, 1 H, J1 = 8.94 Hz, J2 = 2.46 Hz), 4.32–4.29 (t, 2H, J = 6.72 Hz), 3.85 (s, 3H), 1.84–1.78 (sex, 2H, J = 7.38 Hz), 1.05–1.03 (t, 3H, J = 7.38 Hz). 13CNMR (150 MHz, CDCl3) δ
162.0, 154.7, 132.1, 127.88, 127.85, 117.0, 112.7, 108.2, 102.5, 66.5,
55.7, 22.2, 10.5. Elemental Analysis calculated for C13H15NO3: C, 66.94; H, 6.48; N, 6.00. Found:
C, 66.87; H, 6.44; N; 6.00.
Isopropyl 5-Methoxyindole-2-carboxylate
(23d)
This compound was prepared in a manner
similn class="Chemical">ar to that for 23c except anhydrous i-PrOH was deployed
as the reactive solvent, and the mixture was reacted under conditions
of reflux for 4 d to produce a white powder (415 mg, 53%): mp 140–144
°C. TLC R 0.52
(20% EtOAc/hexanes). 1HNMR (600 MHz, CDCl3)
δ 8.78 (s, 1H), 7.32–7.30 (d, 1H, J =
8.94 Hz), 7.13 (m, 1H), 7.07 (d, 1H, J = 2.34 Hz),
7.00–6.98 (dd, 1 H, J1= 8.94 Hz, J2= 2.4 Hz), 5.30–5.25 (sep, 1H, J = 6.3 Hz), 3.85 (s, 3 H), 1.39–1.38 (d, 6H, J = 6.3 Hz). 13CNMR (150 MHz, CDCl3) δ 161.4, 154.7, 132.0, 128.31, 127.86, 116.8, 112.7, 108.0,
102.5, 68.5, 55.7, 22.0. Elemental Analysis calculated for C13H15NO3: C, 66.94; H, 6.48; N, 6.00. Found:
C, 66.80; H, 6.42; N; 6.16.
POCl3 (0.75 mL, 8 mmol) and
n class="Chemical">DMF (2
mL) were stirred at 0 °C for 10 min. To this solution was added 23a (516 mg, 2.5 mmol) in DMF (2 mL) dropwise over 10 min.
The reaction was stirred for an additional 30 min, removed from the
ice bath, and warmed to rt. The reaction mixture was heated to 90
°C for 1.5 h and then poured into ice-cold 1 NNaOH (40 mL).
The crude precipitate was filtered, washed with ice-cold water (25
mL), and dried at 40 °C in a vacuum desiccator for 24 h. The
sample was purified by chromatography (40–70% EtOAc/hexanes)
to provide a yellow solid (265 mg, 45%): mp 243–246 °C.
TLC R 0.43 (50% EtOAc/hexanes). 1HNMR (600 MHz, DMSO-d6) δ
12.82 (s, 1H), 10.58 (s, 1H), 7.69–7.68 (d, 1H, J = 2.4 Hz), 7.47–7.45 (d, 1H, J = 9 Hz),
7.05–7.03 (dd, 1H, J1 = 9.6 Hz, J2 = 2.5 Hz, 1H), 3.98 (s, 3H), 3.80 (s, 3H). 13CNMR (150 MHz, DMSO-d6) δ
187.6, 160.6, 156.6, 132.1, 130.9, 125.6, 118.18, 117.28, 114.2, 102.3,
55.3, 52.7. Elemental analysis calculated for C12H11NO4: C, 61.80; H, 4.75, N, 6.01. Found: C, 61.83;
H, 4.85; N, 6.18.
This compound was prepared from 23d (248 mg, 1.06 mmol) in a manner similn class="Chemical">ar to that for 24c to yield a light yellow powder (261 mg, 93%): mp 201–203
°C. TLC R 0.63
(50% EtOAc/hexanes). 1HNMR (600 MHz, DMSO-d6) δ 12.70 (s, 1H), 10.59 (s, 1H), 7.69 (d, 1H, J = 2.46 Hz), 7.48–7.47 (d, 1H, J = 8.94 Hz), 7.05–7.03 (dd, 1H, J1 = 8.94 Hz, J2 = 2.52 Hz), 5.28–5.24
(sep, 1H, J = 6.3 Hz), 3.80 (s, 3H), 1.40–1.39
(d, 6H, J = 6.3 Hz). 13CNMR (150 MHz,
DMSO-d6) δ 187.5, 159.7, 156.6,
132.7, 130.8, 125.6, 118.04, 117.20, 114.2, 102.3, 69.7, 55.3, 21.6.
Elemental analysis calculated for C14H15NO4·0.2 H2O: C, 63.48; H, 5.86, N, 5.29. Found:
C, 63.24; H, 5.73; N, 5.22.
2-Hydroxymethyl-5-methoxyindole
(25).[22,60]
Compound 23b (2.12g, 9.67 mmol) in THF (40
mL) was stirred under n class="Chemical">Ar at 0 °C for 15 min. 2 M LAH in THF (3
eq, 15 mL) was added to the solution dropwise. The reaction mixture
was stirred at rt for 1 h before the solvent was evaporated in vacuo.
The residue was carefully quenched with ice-cold 0.5 NHCl (200 mL)
and then extracted with EtOAc (200 mL). The organic layer was separated
and dried over Na2SO4. The resulting crude residue
was purified by chromatography (30% to 70% EtOAc/hexanes) to yield
a yellow oil. Drying at 40 °C in a vacuum desiccator for 24 h
produced a yellow solid (1.35 g, 79%): mp 85–88 °C (lit.[22] 80–83 °C). TLC R 0.63 (EtOAc). 1HNMR (600
MHz, DMSO-d6) δ 10.81 (s, 1H), 7.20–7.18
(d, 1H, J = 8.7 Hz), 6.95 (d, 1H, J = 2.4 Hz), 6.67–6.65 (dd, 1H, J1 = 8.7 Hz, J2 = 2.46 Hz), 6.18 (t, 1H, J = 1.2 Hz), 5.20–5.18 (t, 1H, J = 5.64 Hz), 4.56- 4.55 (d, 2H, J = 5.58 Hz), 3.72
(s, 3H). 13CNMR (150 MHz, DMSO-d6) δ 153.0, 140.6, 131.1, 128.1, 111.5, 110.3, 101.4,
98.3, 56.8, 55.1; Elemental analysis calculated for C10H11NO2: C, 67.78; H, 6.26; N, 7.90. Found:
C, 67.56; H, 6.23; N, 7.89.
2-Hydroxymethyl-5-methoxyindole-O-Acetate
(26)
Compound 25 (3.05 mmol, 540
mg), triethylamine (3.36
mmol, 0.47 mL), and n class="Chemical">acetic anhydride (3.66 mmol, 0.35 mL) in CH3CN (20 mL) was stirred at rt for 3 h. The solvent was evaporated
in vacuo and partitioned between EtOAc (50 mL) and saturated NaHCO3 (75 mL). The organic layer was separated, washed with brine
(75 mL), and dried over Na2SO4. The residue
was purified by chromatography (10–40% EtOAc/hexanes) to provide
a light-yellow solid (600 mg, 90%): mp 87–88 °C. TLC R 0.28 (20% EtOAc/hexanes). 1HNMR (600 MHz, CDCl3) δ 8.47 (s, 1H), 7.24–7.23
(d, 1H, J = 8.8 Hz), 7.05–7.04 (d, 1H, J = 2.4 Hz), 6.88–6.86 (dd, 1H, J1 = 8.82 Hz, J2 = 2.5 Hz),
6.46–6.45 (d, 1H, J = 1.6 Hz), 5.20 (s, 2H),
3.84 (s, 3H), 2.10 (s, 3H). 13CNMR (150 MHz, CDCl3) δ 172.3, 154.3, 133.6, 131.7, 127.9, 113.3, 111.9,
103.7, 102.3, 59.8, 55.8, 21.0. Elemental Analysis calculated for
C12H13NO3: C, 65.74; H, 5.98; N,
6.39. Found: C, 65.91; H, 5.86; N, 6.37.
POCl3 (0.6 mL, 2.28 mmol) in
n class="Chemical">DMF (2 mL) was
stirred for 10 min at 0 °C. Compound 26 (470 mg,
2.14 mmol) in DMF (2 mL) was added dropwise over 10 min. The solution
continued to stir at 0 °C for an additional 10 min and then warmed
to rt, after which it was stirred for an additional 45 min. The mixture
was poured into an ice-cold solution of 1 NNaOH (60 mL). The aqueous
mixture was extracted with EtOAc (75 mL × 2) and the latter dried
over Na2SO4 to provide a crude brown solid which
was purified by chromatography (0–5% MeOH/DCM) to yield an
off-white solid (348 mg, 66%): mp 195–197 °C. TLC R 0.57 (3% MeOH/DCM). 1HNMR (600
MHz, DMSO-d6) δ 11.98 (s, 1H), 10.07
(s, 1H), 7.57 (d, 1H, J = 2.64 Hz), 7.35–7.33
(d, 1H, J = 8.7 Hz), 6.83–6.81 (dd, 1H, J1 = 8.76 Hz, J2 =
2.52 Hz), 5.73–5.71 (t, 1H, J = 5.58 Hz),
4.96–4.95 (d, 2H, J = 5.52 Hz), 3.77 (s, 3H). 13CNMR (150 MHz, DMSO-d6) δ
184.1, 155.6, 151.2, 130.2, 126.4, 112.80, 112.52, 112.34, 102.3,
55.25, 55.16. Elemental analysis calculated for C11H11NO3: C, 64.38; H, 5.40; N, 6.83. Found: C, 64.18;
H, 5.25; N, 6.73.
5-Methoxyindole-2-carboxaldehyde (28)
Compound 25 (100 mg, 0.56 mmol) and MnO2 (490
mg, 5.6 mmol) in n class="Chemical">EtOAc (5 mL) were heated to reflux for 24 h. The
mixture was filtered over Celite, and the filtrate was concentrated
in vacuo and purified by chromatography (0% to 10% EtOAc/hexanes)
to yield a pale-yellow powder (75 mg, 76%): mp 143–144 °C
(lit.[61] 140–141 °C). TLC R 0.28 (20% EtOAc/hexanes). 1HNMR (600 MHz, CDCl3) δ 9.81 (s, 1H), 9.05
(s, 1H), 7.36–7.34 (d, 1H, J = 12 Hz), 7.20–7.19
(m, 1H), 7.12–7.11 (d, 1H, J = 6 Hz), 7.09–7.07
(dd, 1H, J1 = 9 Hz, J2 = 2.5 Hz), 3.86 (s, 3H). 13CNMR (150 MHz,
CDCl3) δ 181.8, 155.0, 136.3, 133.4, 127.7, 119.4,
114.15, 113.36, 102.8, 55.7. Elemental Analysis calculated for C10H9NO2: C, 68.56; H, 5.18; N, 8.00.
Found: C, 68.42; H, 5.34; N, 7.85.
Ethyl 3-(5-Methoxyindol-2-yl)-2-propenolate
(29)
To compound 28 (603 mg, 3.44
mmol) in THF
(25 mL) was added (n class="Chemical">carbethoxymethylene)triphenylphosphorane
(1.8 g, 5.16 mmol). The solution was stirred at rt for 20 h. The solvent
was evaporated in vacuo, and the residue was dissolved in DCM (50
mL), washed with brine (50 mL) and then dried over Na2SO4. The resulting material was purified by chromatography (10%
to 40% EtOAc/hexanes) to provide a pale-yellow powder (715 mg, 85%):
mp 138–142 °C (lit.[22] 136–139
°C). TLC R 0.43
(30% EtOAc/hexanes). 1HNMR (600 MHz, CDCl3)
δ 8.35 (s, 1H), 7.67–7.64 (d, 1H, J =
16.02 Hz), 7.24 (s, 1H), 7.04–7.03 (d, 1H, J = 2.34 Hz), 6.94–6.92 (dd, 1H, J1 = 8.82 Hz, J2 = 2.4 Hz), 6.74 (d, 1H, J = 1.62 Hz), 6.22–6.20 (d, 1H, J = 16.02 Hz), 4.30–4.26 (q, 2H, J = 7.14
Hz), 3.84 (s, 3H), 1.36–1.33 (t, 3H, J = 7.08
Hz). 13CNMR (150 MHz, CDCl3) δ 167.3,
154.9, 134.69, 134.23, 133.33, 129.2, 115.97, 115.53, 112.3, 108.8,
102.5, 60.9, 56.0, 14.7. Elemental Analysis calculated for C14H15NO3•0.15 CH2Cl2: C, 65.87; H, 5.98; N, 5.43. Found: C, 66.07; H, 6.04; N, 5.45.
2-Hydroxypropyl-5-methoxyindole (30)
Compound 29 (631 mg, 2.57 mmol) was dissolved in THF (20 mL) and stirred
at 0 °C under n class="Chemical">Argon. 2 M LAH in THF (3 eq, 15 mL) was added dropwise.
The resulting mixture was warmed to rt for 1 h, and volatiles were
evaporated in vacuo. The residue was carefully quenched with ice-cold
0.5 NHCl (200 mL) and then extracted with EtOAc (200 mL). The organic
layer was separated and dried over Na2SO4. The
resulting crude oil was purified by chromatography (30% to 70% EtOAc/hexanes)
to provide a crude yellow solid, which demonstrated ∼85% purity
based on 1HNMR. The impurity was determined to be a partially
reduced alkene intermediate. The yellow solid was further reacted
by dissolving in EtOAc (10 mL) and MeOH (10 mL) in a glass hydrogenation
flask. 10% Pd/C (58 mg, 10% w/w) was added and the sample was hydrogenated
for 4 h at 35 psi of H2 to ensure complete reduction. The
mixture was filtered over Celite and purified by chromatography (20%
to 70% EtOAc/hexanes) to yield a light-yellow solid (216 mg, 41%):
mp 85–88 °C (lit.[22] 62–65
°C). TLC R 0.22
(50% EtOAc/hexanes). 1HNMR (600 MHz, CDCl3)
δ 8.07 (s, 1H), 7.18–7.17 (d, 1H, J =
8.7 Hz), 7.01 (d, 1H, J = 2.4 Hz), 6.78–6.77
(dd, 1H, J1 = 8.7 Hz, J2 = 2.46 Hz), 6.19–6.18 (d, 1H, J = 1.98 Hz), 3.84 (s, 3H), 3.75–3.73 (t, 2H, J = 6.06 Hz), 2.87–2.85 (t, 2H, J = 7.2 Hz),
1.97–1.94 (quin, 2H, J = 6.12 Hz). 13CNMR (150 MHz, CDCl3) δ 154.2, 140.0, 131.2, 129.3,
111.16, 111.04, 102.1, 99.7, 62.3, 56.0, 31.9, 24.9. Elemental analysis
for C12H15NO2: C, 70.22; H, 7.37;
N, 6.82. Found: C, 70.10; H, 7.23; N, 6.74.
2-Hydroxypropyl-5-methoxyindole-O-acetate
(31)
To compound 30 (300 mg, 1.46
mmol) in CH3CN (10 mL) was added TEA (0.22 mL, 1.61 mmol)
and n class="Chemical">acetic anhydride
(0.18 mL, 1.75 mmol). The solution was stirred at rt for 20 h. Volatiles
were evaporated in vacuo, and the residue was dissolved in EtOAc (50
mL), washed with saturated NaHCO3 (75 mL) and brine (50
mL). The organic layer was separated, dried over Na2SO4, and purified by chromatography (0% to 50% EtOAc/hexanes
to yield a pale-yellow solid (190 mg, 53%): TLC R 0.18 (17% EtOAc/hexanes). mp 86–90
°C. 1HNMR (600 MHz, CDCl3) δ 7.98
(s, 1H), 7.20–7.19 (d, 1H, J = 8.7 Hz), 7.01
(d, 1H, J = 2.46 Hz), 6.79–6.77 (dd, 1H, J1 = 8.7 Hz, J2 =
2.4 Hz), 6.19 (d, 1H, J = 2.1 Hz), 4.18–4.16
(t, 2H, J = 6.36 Hz), 3.84 (s, 3H), 2.82–2.80
(t, 2H, J = 7.38 Hz), 2.07 (s, 3H), 2.06–2.02
(quin, 2H, J = 6.36 Hz). 13CNMR (150
MHz, CDCl3) δ 171.6, 154.4, 139.4, 131.2, 129.4,
111.29, 111.25, 102.2, 100.0, 63.8, 56.1, 28.7, 24.9, 21.2. Elemental
analysis calculated for C14H17NO3: C, 68.00; H, 6.93; N, 5.66. Found: C, 67.95; H, 6.91; N, 5.65.
POCl3 (0.17 mL, 1.82 mmol) and n class="Chemical">DMF
(1.5 mL) stirred at 0 °C for 10 min. Compound 31 (150 mg, 0.61 mmol) in DMF (2 mL) was added dropwise at 0 °C
for an additional 10 min. The sample was removed from the ice-bath
and reacted for 2 h at rt. The reaction mixture was poured into ice-cold
H2O (15 mL) and 1 NNaOH was added dropwise until a pH
of 10 was determined by pH paper (10 mL). The flask was refrigerated
for 20 min to facilitate the formation of a precipitate. The precipitate
was collected, washed with ice-cold H2O (20 mL) and dried
at 40 °C in a vacuum desiccator for 24 h to provide a cream-colored
solid (108 mg, 64%): mp 170–174 °C. TLC R 0.45 (4:1 EtOAc/hexanes). 1HNMR (600
MHz, DMSO-d6) δ 11.91 (s, 1H), 10.03
(s, 1H), 7.58 (d, J = 2.52 Hz, 1H), 7.30–7.29
(d, J = 8.76 Hz, 1H), 6.82–6.80 (dd, J1 = 8.7 Hz, J2 =
2.52 Hz, 1H), 4.04–4.02 (t, J = 6.42 Hz, 2H),
3.77 (s, 3H), 3.13–3.10 (t, J = 7.5 Hz, 2H),
2.06–2.01 (quin, J = 6.42 Hz, 2H), 1.97 (s,
3H). 13CNMR (150 MHz, DMSO-d6) δ 184.0, 170.4, 155.5, 151.3, 130.2, 126.3, 113.5, 112.26,
112.15, 102.3, 63.0, 55.3, 28.5, 22.2, 20.7. Elemental Analysis calculated
for C15H17NO4·0.19H2O: C, 64.64; H, 6.28; N, 5.03. Found: C, 64.25; H, 6.49; N, 5.04.
Cell Proliferation
The humann class="CellLine">U251 glioblastoma cell
line was obtained from the DCT Tumor Repository (National Cancer Institute,
Fredrick, MD). Cells were maintained in Dulbecco’s modified
Eagle medium supplemented with 10% fetal bovine serum, as described
previously.[16] The effects of compounds
on cell growth were assessed using the sulphorhodamine B (SRB) colorimetric
assay,[62] as described previously.[17] Cells were seeded at an initial density of 2000
cells per well in 96-well plates, with four replicate wells for each
drug concentration. All test compounds were dissolved in DMSO and
serially diluted in DMSO so that the desired final drug concentrations
could be achieved by making a 1/1000 dilution into the culture medium.
One day after plating, four wells were assayed to establish a predrug
(time-0) baseline. At the same time, fresh medium containing test
compounds was added to the remaining wells. Control wells received
DMSO alone. SRB assays were performed at a 48 h end point. To minimize
the impact of potential variations in stability among the compounds,
medium with test compound was replenished after the first 24 h. The
concentration of each compound producing 50% growth inhibition (GI50) relative to the control without drug was calculated as
described in the NCI-60human cell line screening protocol (http://dtp.nci.nih.gov/branches/btb/ivclsp.html).
Cell Morphology
For acquisition of phase-contrast images
of live cells, U251 cells were seeded on 35 mm diameter plastic culture
dishes at 105 cells per dish. After 1 day, fresh medium
with test compound was added. Images were obtained at the indicated
time intervals after addition of drug, using an Olympus IX70 inverted
microscope equipped with a DP-80 digital camera and CellSens 1.9 imaging
softwn class="Chemical">are (Olympus America, Center Valley, PA).
Cell Cycle
Analysis
Guava Cell Cycle Reagent was purchased
from EMD Millipore Corporation, Haywn class="Chemical">ard, CA. U251 cells were seeded
at 200 000 cells per 35 mm dish 1 day prior to addition of
test compounds. Cell cycle analyses were performed 24 h after addition
of test compounds, using a Guava Personal Cytometer (EMD Millipore)
according to the manufacturer’s protocol for the Guava Cell
Cycle Reagent. CytoSoft 2.1.4 software was used to analyze the resulting
DNA histograms and calculate the percentage of cells in each phase
of the cell cycle, based on a total of 5000 events per sample. Statistical
significance of differences between cells treated with specific compounds
and control (DMSO-treated) cells was assessed using Student’s
two tailed t test.
Immunofluorescence Microscopy
U251 cells were seeded
on glass coverslips in 60 mm dishes at 350 000 cells per dish.
One day after plating, fresh medium was added with compounds at the
indicated concentration. Cells were fixed with ice-cold n class="Chemical">methanol,
and α-tubulin was detected by immunofluorescence microscopy,
using a primary mouse monoclonal antibody (Sigma Chemical Co., St.
Louis, MO) followed by Alexa Fluor 568-labeled goat antimouse IgG
(Life Technologies, Grand Island, NY). After washing away excess antibodies,
nuclear DNA was stained for 5 min with 300 nM 4′,6-diamidino-2-phenyl-indole
(DAPI) (Sigma Chemical Co.). Fluorescent images were obtained on an
Olympus IX70 inverted microscope.
Tubulin Fractionation Assay
One day after seeding U251
cells at 1.4 × 106 cells per 10 cm dish, cells were
treated for 4 h with the indicated concentration of drugs. At the
end of the incubation, the medium was aspirated, and the cells were
quickly scraped into 700 μL of lysis buffer consisting of 100
mM n class="Chemical">PIPES, pH 6.9, 5 mM MgCl2, 1 mM EGTA, 30% glycerol,
0.1% NP-40, 0.1% Triton X-100, 0.1% Tween-20, 0.1% 2-mercaptoethanol,
2 mM GTP, and protease inhibitor cocktail (Cystoskeleton Inc., Denver,
CO). The lysis buffer was briefly prewarmed to 37 °C prior to
addition to the cells. 600 μL of the lysate was transferred
to a prewarmed tube and centrifuged at 100000g for
1 h at 37 °C. The resulting supernatant solution contained the
nonpolymerized tubulin and the pellet contained the polymerized tubulin.
0.24% of each fraction was separated on a 10% SDS-polyacrylamide gel,
and tubulin was detected by immunoblot analysis using a mouse monoclonal
antibody against α-tubulin (Sigma) using established methods.[14] Chemiluminescent signals from the tubulin immunoblots
were quantified using an Alpha Innotech FluorChem HD2 imaging system
(San Leandro, CA). Statistical significance of differences between
cells treated with specific compounds and control (DMSO-treated) cells
was assessed using Student’s two tailed t test.
Authors: Christine Dyrager; Malin Wickström; Maria Fridén-Saxin; Annika Friberg; Kristian Dahlén; Erik A A Wallén; Joachim Gullbo; Morten Grøtli; Kristina Luthman Journal: Bioorg Med Chem Date: 2011-03-10 Impact factor: 3.641
Authors: Stuart A Grossman; Xiaobu Ye; Steven Piantadosi; Serena Desideri; Louis B Nabors; Myrna Rosenfeld; Joy Fisher Journal: Clin Cancer Res Date: 2010-04-06 Impact factor: 12.531
Authors: P Skehan; R Storeng; D Scudiero; A Monks; J McMahon; D Vistica; J T Warren; H Bokesch; S Kenney; M R Boyd Journal: J Natl Cancer Inst Date: 1990-07-04 Impact factor: 13.506
Authors: Danielle D Jandial; Christopher A Blair; Saiyang Zhang; Lauren S Krill; Yan-Bing Zhang; Xiaolin Zi Journal: Curr Cancer Drug Targets Date: 2014 Impact factor: 3.428
Authors: Shengnan Du; Jeffrey G Sarver; Christopher J Trabbic; Paul W Erhardt; Allen Schroering; William A Maltese Journal: Cancer Chemother Pharmacol Date: 2018-11-13 Impact factor: 3.333
Authors: Christopher J Trabbic; Sage M George; Evan M Alexander; Shengnan Du; Jennifer M Offenbacher; Emily J Crissman; Jean H Overmeyer; William A Maltese; Paul W Erhardt Journal: Eur J Med Chem Date: 2016-06-13 Impact factor: 6.514
Authors: Delia Preti; Romeo Romagnoli; Riccardo Rondanin; Barbara Cacciari; Ernest Hamel; Jan Balzarini; Sandra Liekens; Dominique Schols; Francisco Estévez-Sarmiento; José Quintana; Francisco Estévez Journal: J Enzyme Inhib Med Chem Date: 2018-12 Impact factor: 5.051
Figure 1
Scheme 1
Scheme 4
Scheme 2
Scheme 3
Scheme 5
Scheme 6
Scheme 7
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9