Analogues and derivatives of oncrasin-1, a novel inhibitor of the C-terminal domain of RNA polymerase II and their antitumor activities

Article abstract of DOI:10.1021/jm101417n

To optimize the antitumor activity of oncrasin-1, a small molecule RNA polymerase II inhibitor, we evaluated 69 oncrasin-1 analogues for their cytotoxic activity against normal human epithelial cells and K-Ras mutant tumor cells. About 40 of those compoun

Full text of DOI:10.1021/jm101417n

ARTICLE
pubs.acs.org/jmc
Analogues and Derivatives of Oncrasin-1, a Novel Inhibitor of the
C-Terminal Domain of RNA Polymerase II and Their Antitumor
Activities
Shuhong Wu,† Li Wang,† Wei Guo,† Xiaoying Liu,† Jinsong Liu,‡ Xiaoli Wei,§ and Bingliang Fang*,†
†Department of Thoracic and Cardiovascular Surgery and ‡Department of Pathology, The University of Texas MD Anderson Cancer
Center, Houston, Texas 77030, United States
§Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China
ABSTRACT: To optimize the antitumor activity of oncrasin-1, a small molecule RNA
polymerase II inhibitor, we evaluated 69 oncrasin-1 analogues for their cytotoxic activity against
normal human epithelial cells and K-Ras mutant tumor cells. About 40 of those compounds were
as potent as or more potent than oncrasin-1 in tumor cells and had a minimal cytotoxic effect on
normal cells. Structureꢀactivity relationship analysis revealed that most of the active compounds
contained either a hydroxymethyl group or an aldehyde group as a substitute at the 3-position of
the indole. Both electron-donating and electron-withdrawing groups in the benzene ring were
well tolerated. The hydroxymethyl compounds ranged from equipotent with to 100 times as
potent as the corresponding aldehyde compounds. We tested three active analogues' effect on
RNA polymerase phosphorylation and found that they all inhibited phosphorylation of the C-terminal domain of RNA polymerase
II, suggesting that the active compounds might act through the same mechanisms as oncrasin-1.
’ INTRODUCTION
Similarly, oncogene-transformed cells, such as c-Myc-trans-
formed cells, were reported to be more sensitive than their
normal counterparts to RNA polymerase II inhibitors, regardless
of p53 status, suggesting that RNA polymerase II may serve as a
therapeutic target for anticancer therapy.12 Indeed, anticancer
agents such as the pan-cyclin-dependent kinase (CDK) inhibi-
tors flavopiridol and seliciclib elicit their antitumor activity by
inhibiting RNA polymerase II function,13ꢀ15 leading to suppres-
sion of the expression of short-lived antiapoptotic proteins such
as Mcl-1, XIAP, Bcl-XL, and survivin.16ꢀ18 Taken together, these
findings indicate that agents that selectively inhibit RNA poly-
merase II function will likely be safe and tolerable and may have
anticancer efficacy.
We recently reported a novel anticancer agent, designated
oncrasin-1, that was identified through synthetic lethality screen-
ing on isogenic human ovarian epithelial cells with or without
oncogenic Ras expression.8 Molecular characterization revealed
that oncrasin-1 induced apoptosis in a subset of cancer cell lines
associated with induction of coaggregation of protein kinase Cι
and RNA splicing factors in the nucleus and suppression of
phosphorylation of the C-terminal domain (CTD) of the largest
subunit of eukaryotic RNA polymerase II.19 CTD phosphoryla-
tion is known to be essential for efficient transcription elongation
and recruitment of mRNA processing factors, including the capping
enzyme and splicing factors required for efficient processing of RNA
transcripts.20ꢀ26 Inhibiting CTD phosphorylation will ultimately
disrupt RNA polymerase II function and promote cell death.16
Genetic and epigenetic changes in cancer cells that lead to
functional alterations in several key signaling pathways are
believed to be the driving force behind oncogenesis and malig-
nant evolution of cancers.1,2 Those functional changes in signal-
ing pathways have recently been exploited for the development
of anticancer therapeutics. Small molecules directly inhibiting
critical nodes in oncogenic signaling networks—such as erlotinib
and gefitinib against epidermal growth factor receptor,3 imatinib
against BCR-Abl fusion protein,4 and sorafenib against Raf kinase
and vascular endothelial growth factor receptor5—have already
been successfully used for the treatment of various cancers in
humans. Small molecules that selectively induce cell death
through synthetic lethality in a subset of deregulated oncogene-
or mutant tumor suppressor gene-transformed malignant cells
but not in their normal counterparts have also been exploited as
tumor-selective anticancer agents.6ꢀ8 Evidence has demon-
strated that functional changes caused by gain-of-function muta-
tions in oncogenes or loss-of-function mutations in tumor
suppressor genes may alter cellular signaling or metabolism,
rendering the mutant cells more susceptible to a functional
change in another gene or rendering the mutant cells susceptible
to cell death due to synthetic lethality.9
The advantage of synthetic lethality-mediated anticancer
therapy is that it provides lethal selectivity against cancer cells
and the opportunity to eliminate malignant cells by indirectly
targeting cancer-driving molecules that otherwise cannot be
targeted. For example, defects in BRCA1 and BRCA2 genes in
some breast and ovary cancer cells make them highly sensitive to
small molecule inhibitors of poly (ADP-ribose) polymerase.10,11
Received: November 3, 2010
Published: March 28, 2011
r
2011 American Chemical Society
2668
dx.doi.org/10.1021/jm101417n J. Med. Chem. 2011, 54, 2668–2679
|

Journal of Medicinal Chemistry
ARTICLE
Scheme 1. General Approaches for the Syntheses of Oncra-
sin-1 Analoguesa
products were confirmed by nuclear magnetic resonance
(NMR) spectrum analyses. For compounds that had purity
g95%, antitumor activity was tested in cultured cells.
Antitumor Activity and Selectivity. Oncrasin-1 was origin-
ally identified through synthetic lethality screening on isogenic
T29 immortalized normal human ovary epithelial cells and a T29
tumorigenic subclone derived from transformation with mutant
K-Ras (T29Kt1).8,29 Later testing showed that several lung
cancer cell lines harboring K-Ras mutations were highly sensitive
to oncrasin-1, including H460.8 Therefore, we used T29,
T29Kt1, and H460 cell lines to evaluate the cytotoxicity and
cancer cell selectivity of oncrasin-1 analogues and derivatives.
Cells were treated with each compound at doses ranging from
10ꢀ4.5 (31.6 μM) to 10ꢀ8 M (10 nM) for 3 days, and cell viability
was determined by using a sulforhodamine B assay. Cells treated
with 1% dimethyl sulfoxide were used as controls. The dose that
caused a 50% reduction in the number of surviving cells as
compared with the number of surviving cells in the control group
(the IC50 value) was then calculated for each compound tested.
As shown in Table 1, the IC50 values for 70 compounds
(including oncrasin-1) tested on T29, T29Kt1, and H460 cells
ranged from 0.01 to >31 μM. About 40 compounds showed
antiproliferative activity in T29Kt1 and H460 cells with IC50
values at low micromole (e5 μM) or nanomole levels (Figure 1).
For most of those approximately 40 active compounds, the IC50
for T29 cells was greater than 31.6 μM, the highest concentration
tested, suggesting that those compounds were relatively nontoxic
for T29 cells and, like oncrasin-1, were active against both H460
cells and T29Kt1 cells. In a few cases, however, a compound was
active against H460 cells but not active against T29Kt1 cells.
Most of the active compounds had IC50 values less than or
equivalent to those of oncrasin-1, which had IC50 values of
>31 μM for T29 cells, 2.51 μM for T29Kt1 cells, and 0.25 μM for
H460 cells.
StructureꢀActivity Relationships. A structureꢀactivity re-
lationship analysis based on IC50 values (Table 1) showed that
variations in substitutions of the benzyl group did not have a
dramatic impact on the cytotoxicity and selectivity of the
oncrasin-1 analogues and derivatives. Both electron-donating
and electron-withdrawing groups were well tolerated. Various
substitutions of the benzyl group may have changed the IC50
value in H460 and T29Kt1 cells; however, the changes were
quantitative rather than qualitative. Replacement of the benzyl
group with an isopropyl group (compound 22) resulted in
complete or partial loss of activity in H460 and T29Kt1 cells.
On the other hand, the linker between the benzyl group and
the 1-H indole was not very strict. Although most of the com-
pounds tested had one carbon atom between the indole and the
benzene group, a compound with the linker of ꢀ(CH2)2ꢀOꢀ
(compound 15) retained its activity and selectivity for H460 and
T29Kt1 cells. Those results suggested that an aryl substitute at
the 1-position of the 1-H indole is preferred for antitumor
activity, although the linker between the benzene ring and the
1-H indole is not very strict.
a Each synthesis step is described in detail in the Experimental Section.
Compounds identified by library screening often need to be
optimized before their possible clinical application can be
explored. To optimize oncrasin-1, we have synthesized several
oncrasin-1 analogues and derivatives and tested their antitumor
activity and selectivity in cultured cell lines. Here, we report the
structureꢀactivity relationships of these oncrasin-1 analogues
and their derivatives.
’ RESULTS
Synthesis of Oncrasin-1 Analogues and Derivatives. The
lead compound, oncrasin-1, was previously reported to induce
antitumor activity in a subset of cancer cells harboring K-Ras
mutations and to inhibit phosphorylation of the CTD of RNA
polymerase II;8,19 oncrasin-1 contains an indole structure similar
to indole-3-carbinol and a benzyl ring attached to the N atom of
the indole. Indole-3-carbinol is a natural constituent of crucifer-
ous vegetables that has been tested for cancer prevention and
therapy.27,28 However, in our initial studies, we found that indole-
3-carbinol did not have observable antitumor activity in cancer
cells that were highly susceptible to oncrasin-1,8 suggesting that a
benzyl group linked to the indole is required for the antitumor
activity observed in oncrasin-1. Therefore, most analogues
synthesized and tested here contained an indole structure with
a benzyl group linked to the N atom of the indole. Occasionally,
other molecules were used to replace the benzyl group in the
reaction.
The general approaches for the syntheses of oncrasin-1
analogues are presented in Scheme 1. Briefly, indole-3-carbox-
aldehyde or its analogues were reacted with a benzyl halide under
alkali catalysts at room temperature. The products, which were
purified on a silica gel or a neutral aluminum C18 column with a
CombiFlash Rf-200 system (Teledyne), were either tested for
antitumor activity or reduced, oxidized, or otherwise modified to
obtain various derivatives as listed in Table 1. According to the
substitutes on the 3-position of the indole ring, the derivatives
can be subgrouped as carboxaldehyde, methanol, hydroxyalkyl,
carboxylic acid, ester, amine, amide, and others. The purity and
molecular weight of the final products were determined by high-
performance liquid chromatographyꢀmass spectrometry
(HPLC/MS/UV230). The chemical structures of the final
Most of the active compounds contained either an aldehyde
group or a hydroxymethyl group as a substitute at the 3-position
of the indole. In general, a compound with 3-methanol was more
active than its 3-carbaldehyde counterpart. In H460 cells, most
hydroxymethyl derivatives (compounds 30ꢀ55) were equipo-
tent with to six times as potent as the corresponding aldehyde
compounds (compounds 1ꢀ22), whereas in T29K cells, most
hydroxymethyl derivatives were 1ꢀ100 times as potent as the
2669
dx.doi.org/10.1021/jm101417n |J. Med. Chem. 2011, 54, 2668–2679

Journal of Medicinal Chemistry
ARTICLE
Table 1. Analogues and Derivatives of Oncrasin-1 and Their Antitumor Activities (IC50, μM)
2670
dx.doi.org/10.1021/jm101417n |J. Med. Chem. 2011, 54, 2668–2679

Journal of Medicinal Chemistry
ARTICLE
Table 1. Continued
Figure 1. IC50 values of oncrasin-1 and its analogues in T29, T29Kt1, and H460 cells. All IC50 values of g31.6 μM were recorded as 31.6 μM.
corresponding aldehyde compounds (Figure 1). These trends were
not dependent on the site or nature (electron donating or with-
drawing) of substitution on the benzyl group at the 1-position of
indole. Substituting R1 with a ketone (compounds 26ꢀ29), a
secondary alcohol (compound 58), a methyl (compound 66), or a
carboxylic acid (compounds 62 and 63) led to dramatic reduction
or complete loss of activity. Moreover, extension of the R1
hydroxymethyl chain by one carbon (compound 56) or two
carbons (compound 57) also resulted in dramatic reduction or
complete loss of activity in both H460 and T29Kt1 cells, suggesting
that the substitute in the indole-3 position is critical for the antitumor
activity of this group of compounds. Methylation of the primary
alcohol also resulted in a dramatic loss of activity (compound 59).
Nevertheless, acylation of the hydroxymethyl group (compound
60) produced a compound equipotent to compound 35.
The effects of substitutes in other positions of the indole
varied, depending on the position and substitute. Substitution of
the indole at the 5-position or 6-position (compounds 51ꢀ55)
was well tolerated; 5-F substitution (compounds 51 and 55)
produced the best potency.
conditions. At pH 2.0, a large portion of those compounds
formed dimers within about 30 min (Figure 2). A similar dimer
formation at an acidic condition has been reported for indole-3-
carbinol,30 which is major part of hydroxymethyl compounds
tested here. Nevertheless, hydroxymethyl compounds were
stable at pH 4, 6, 7.4, and 9. In contrast, aldehyde compounds
were stable at both pH 2.0 and 7.4. We synthesized compounds
68 and 69 and evaluated antitumor activity of the dimers. The
results showed that dimerization of hydroxymethyl compounds
led to complete loss of antitumor activity (compounds 68 and
69). These findings suggested that a specific formulation, such as
an enteric-coated formulation, would be required for orally
administered hydroxymethyl compounds to be active in vivo.
We also analyzed whether selective cell killing by an active
compound is caused by a difference in bioavailability between
sensitive and resistance cell lines. For this purpose, we treated
T29 and T29Kt1 cells with 10 μM compound 34. Because
T29Kt1 cells started to die at about 6 h after the treatment, we
analyzed the cellular content of compound 34 overtime up to 5 h
after the treatment. The result showed that at all time points
tested, the levels of compound 34 in T29 cells were twice as that
in T29Kt1 cells (Figure 2D). This result suggested that the
Stability of Hydroxymethyl Compounds. We found that
hydroxymethyl compounds were not stable under acidic
2671
dx.doi.org/10.1021/jm101417n |J. Med. Chem. 2011, 54, 2668–2679

Journal of Medicinal Chemistry
ARTICLE
Figure 2. Stability of a representative hydroxymethyl analogue at different pH. (A) Results of HPLC analyses after incubation of compound 34 at pH 2.0
and pH 7.4 for the indicated times. (B) Structure of the tested compound (compound 34) and its dimerized form (compound 68). (C) Percentage of
compound 34 remaining in solution after incubation at pH 2.0ꢀ9 for the indicated times. (D) Levels of compound 34 in T29 and T29Kt1 cells overtimes
after treatment with 10 μM compound 34.
Figure 3. Antitumor activity of three active oncrasin-1 analogues, compounds 34, 35, and 50, in lung cancer cell lines. (A) Dose-dependent antitumor
activity. Four lung cancer cell lines (H460, H157, H1299, and H322) were treated with various concentrations of the compounds ranging from 0.031 to
31 μM. Cell viability was determined 3 days after treatment by using sulforhodamine B assays. Control cells were treated with solvent (dimethyl
sulfoxide), and their value was set as 1. The values shown are the means from a quadruplicate assay. (B) Western blot analysis. Lung cancer cells were
treated with the compounds at a concentration of 1 μM for 24 h. Phosphorylation of the CTD or the largest subunit of RNA polymerase II was
determined with H5 pPol II antibody. β-Actin was used as the loading control. C, control; T, treatment.
2672
dx.doi.org/10.1021/jm101417n |J. Med. Chem. 2011, 54, 2668–2679

Journal of Medicinal Chemistry
ARTICLE
resistance observed in T29 cells was not caused by low bioavail-
ability or metabolic instability of the compound in the cells.
Effect on Phosphorylation of the CTD of RNA Polymerase
II. Our initial mechanistic characterization on oncrasin-1-induced
antitumor activity revealed that oncrasin-1 suppressed phosphor-
ylation of the CTD of the largest subunit of RNA polymerase II in
sensitive cancer cell lines but not in resistant cancer cell lines.19
Phosphorylation of the CTD is essential for efficient mRNA
transcription processing, including capping and splicing.20ꢀ26
Inhibition of CTD phosphorylation is reported to promote cell
death in transformed cells and cancer cells.12,16
To determine whether active oncrasin-1 derivatives also
inhibited CTD phosphorylation, we tested the effect of three
active oncrasin-1 analogues (compounds 34, 35, and 50) in two
oncrasin-1-sensitive (H157 and H460) and two oncrasin-1-
resistant (H1299 and H322) lung cancer cell lines. The effect
of these three active compounds on cell viability at 3 days after
treatment is shown in Figure 3A. Both H157 and H460 cells were
susceptible to the three oncrasin-1 analogues; IC50 values were
e0.2 μM. In contrast, both H1299 and H322 cells were resistant
to the oncrasin-1 analogues; IC50 values were g13 μM. Analysis
of CTD phosphorylation of cells treated with the oncrasin-1
analogues at a concentration of 1 μM for 12 h showed that all
three compounds dramatically suppressed CTD phosphoryla-
tion in H157 and H460 cells but not in H1299 and H322 cells
(Figure 3B). These results demonstrated that the active oncrasin-
1 analogues, like oncrasin-1, effectively suppressed CTD phos-
phorylation in sensitive cancer cell lines but not in resistant
cancer cell lines.
agents also function as inhibitors of RNA polymerase II. The
CTD of the largest subunit of eukaryotic RNA polymerase II
contains multiple heptapeptide repeats of the sequence
YSPTSPS, 26 in the yeast CTD, and 52 in the mammalian
CTD.31 The serine and threonine in the CTD exhibit variable
phosphorylation, which is required for efficient transcription
elongation and recruitment of mRNA processing factors, includ-
ing the capping enzyme and splicing factors required for efficient
processing of RNA transcripts.24,26,32 The CTD is known
to be phosphorylated by three major CDKs, CDK7/cyclin
H (TFIIH),33,34 CDK8/cyclin C,35,36 and CDK9/cyclin T
(P-TEFb).37 In addition to the CDKs, there are other protein
kinases that also efficiently phosphorylate the CTD, includ-
ing the DNA-dependent protein kinase,38 the extracellular
signal-regulated kinase,39 and the c-abl tyrosine kinase.40,41
RNA polymerase II enters the assembling transcription complex
with its CTD unphosphorylated (IIa form). Phosphorylation of
CTD by CDK7 and CDK9 converts polymerase II to its phos-
phorylated form, enabling efficient RNA elongation and
processing.24,26 How oncrasin-1 and its analogues inhibit phos-
phorylation of the CTD remains to be determined. Our previous
study revealed that the direct inhibitory effects of oncrasin-1 on
CDK9 and CDK7 were moderate, suggesting that other protein
kinases or indirect mechanisms might contribute to the inhibi-
tion of CTD phosphorylation by this group of compounds.
Using small molecule inhibitors of RNA polymerase II as
anticancer agents was suggested by Koumenis and Giaccia on the
basis of their observation that an RNA polymerase inhibitor, 5,6-
dichloro-l-/3-D ribofuranosylbenzimidazole, induced apoptosis
more effectively in oncogene-transformed cells than in untrans-
formed cells.12 However, to date, there has been little work in this
area because of the potential toxicity of small molecular RNA
polymerase II inhibitors. Nevertheless, some recent studies have
revealed that certain anticancer agents, including flavopiridol and
seliciclib, elicit their antitumor activity by blocking CTD
phosphorylation,13ꢀ15 providing evidence that inhibiting the
CTD function of RNA polymerase II can be exploited as a novel
anticancer approach. Our study demonstrates that a group of
small molecules could inhibit CTD phosphorylation and induce
antitumor activity in a subset of cancer cells without obvious toxic
effects on normal cells. Those agents may be a starting point for
new anticancer therapeutics.
’ DISCUSSION AND CONCLUSION
We evaluated 69 oncrasin-1 analogues for their antitumor
activity and selectivity in normal and cancerous cell lines. Our
results showed that about 40 compounds had antitumor activity
similar to or greater than that of oncrasin-1 in H460 lung cancer
cells and T29Kt1 ovarian cancer cells. Most of those active
compounds also retained their selectivity against tumorigenic
H460 and T29Kt1 cells as compared with the normal ovarian cell
line T29. Our results suggest that these oncrasin-1 analogues and
derivatives represent a group of small molecules that have an
antitumor spectrum similar to that of oncrasin-1 and possibly
molecular mechanisms similar to that of oncrasin-1. Most active
compounds contain either a hydroxymethyl group or an alde-
hyde group at the indole-3-position, suggesting that the active
compounds may interact with some cellular nucleophiles to
induce antitumor activity. Analysis on intracellular levels of
compound 34 in T29 and T29Kt1 cells showed that the resistant
cells had relatively higher levels of the compound than the
sensitive cells did. Whether this is caused by consumption of
the compound through conjugation with cellular nucleophiles
(glutathione, proteins, or nucleic aids) in the sensitive cells,
either directly or indirectly through metabolites, remains to be
further investigated. It is possible that some metabolic or
biochemical differences between the normal and the tumor cells
may account for the observed selectivity. Nevertheless, a high
level of an active compound in resistant cells suggested that the
resistance is not associated with a membrane pump-mediated
compound efflux mechanism.
’ EXPERIMENTAL SECTION
Chemicals. All reagents were purchased from Sigma-Aldrich or
Chembridge in the highest commercially available quality. Of 69
analogous listed in Table 1, nine compounds (compounds 1, 15, 22,
23, 33, 47, 57, 62, and 65) were obtained from ChemBridge Corp
(San Diego, CA), one (compound 14) was from Sigma-Aldrich
(St. Louis, MO), and one (compound 26, NSC118788) was from the
Developmental Therapeutics Program of the National Cancer Institute.
The other 58 compounds were synthesized in our laboratory. Solvents
were reagent grade, HPLC grade, or MS grade, depending on whether
they were to be used for synthesis or chemical analysis.
Compound Synthesis. The compounds and their intermediates
were synthesized as illustrated in Scheme 1 and are briefly described in
the following paragraphs.
Step A. Sodium hydride (1.1 mmol) dispersed in mineral oil was
added to a solution of 1H-indole-3-carboxaldehyde or analogues
(1.0 mmol) in anhydrous dimethyl sulfoxide at room temperature with
stirring. After the solution was stirred for 1 h, benzyl halide (1.2 mmol)
Our results showed that, like oncrasin-1, all three active
compounds inhibited CTD phosphorylation in sensitive cancer
cells but not resistant cancer cells, demonstrating that those
2673
dx.doi.org/10.1021/jm101417n |J. Med. Chem. 2011, 54, 2668–2679

Journal of Medicinal Chemistry
ARTICLE
was added, and the mixture was stirred continuously for 3ꢀ5 h. The
reaction was stopped by adding iceꢀwater, and the mixture was
extracted with dichloromethane thrice and brined with 10% NaCl
aqueous solution. The organic phase was dried over anhydrous Na2SO4
and filtered, and solvent was removed under reduced pressure. The
residue was dissolved in CHCl3 and separated on a silica gel column with
CHCl3/MeOH. The products were concentrated using a vacuum
evaporator and recrystallized in diethyl ether/n-hexane.
Step B. A product (1.0 mmol) from step A was dissolved in acetone
and treated with KMnO4 (1.9 mmol in water). The resulting mixture
was stirred for 16 h at room temperature. The products were decolored
with 10% H2O2 and filtered, and the residues were removed. Solvent
acetone was reduced to one-third volume under reduced pressure. The
resulting solution was acidified with 2 N HCl to obtain a precipitate. The
product was recrystallized in ethanol.
Step C. A product (1.0 mmol) from step A suspended in alcohol was
reduced with an equimolar amount of sodium borohydride on ice bath.
The mixture was stirred at room temperature for 1ꢀ2 h, and the reaction
was stopped by adding acetone. The mixture was poured into iceꢀwater
and incubated at 4 °C overnight. The precipitate was washed with water
thrice and dissolved in chloroform. The organic phase was dried over
anhydrous Na2SO4. Finally, the products were purified on a silica gel
column with chloroform/MeOH as an eluent.
Step D. A product from step C was modified with aliphatic acid
to form an ester product. The product (5 mmol) was dissolved in
anhydrous dichloromethane completely by stirring. Aliphatic (C2ꢀC8)
carboxylic acid (7.5 mmol), 1,3-dicyclohexylcarbodiimide (5.5 mmol),
and 4-(dimethylamine) pyridine (1 mmol) were added into the solution.
The mixture was stirred at room temperature for 2ꢀ3 h. The product
was filtered through paper film, concentrated by evaporation under
reduced pressure, redissolved in ethyl acetate, and filtered through
Celite521. The solvent was removed with clean nitrogen gas, and the
product was purified on a column of neutral alumina by eluting with
dichloromethane/hexane.
CDCl3): δ 10.05 (m, 1H, HꢀCdO indole), δ 8.36 (m, 1H, C4-indole),
δ 7.75 (m, 1H, C7-indole), δ 7.36ꢀ7.20 (s, 2H, C5, C6-indole and 4H,
phenyl), δ 7.04 (m, 1H, C2-indole), δ 5.36 (s, 2H, NꢀCH2). ESI-MS
m/z 269.9 [M þ H].
1-[(3-Bromophenyl)methyl]-1H-indole-3-carboxaldehyde (4).
Compound 4 was prepared from 1H-indole-3-carboxaldehyde and
1
3-bromobenzyl bromide as described in step A. H NMR (300 MHz,
CDCl3): δ 10.06 (m, 1H, HꢀCdO indole), δ 8.36 (m, 1H, C4-indole),
δ 7.75 (m, 1H, C7-indole), δ 7.48ꢀ7.24 (s, 2H, C5, C6-indole and 4H,
phenyl), δ 7.08 (m, 1H, C2-indole), δ 5.36 (s, 2H, NꢀCH2). ESI-MS
m/z 314.0 [M þ H].
1-[(4-Bromophenyl)methyl]-1H-indole-3-carboxaldehyde (5).
Compound 5 was prepared from 1H-indole-3-carboxaldehyde and
1
4-bromobenzyl bromide as described in step A. H NMR (300 MHz,
CDCl3): δ 10.06 (m, 1H, HꢀCdO indole), δ 8.36 (m, 1H, C4-indole),
δ 7.75 (m, 1H, C7-indole), δ 7.49ꢀ7.24 (s, 2H, C5, C6-indole and 4H,
phenyl), δ 7.08 (m, 1H, C2-indole), δ 5.36 (s, 2H, NꢀCH2). ESI-MS
m/z 314.0 [M þ H].
1-[(3-Trifluoromethylphenyl)methyl]-1H-indole-3-carboxaldehyde
(6). Compound 6 was prepared from 1H-indole-3-carboxaldehyde
1
an 3-trifluoromethylbenzyl chloride in step A. H NMR (300 MHz,
DMSO-d6): δ 9.09 (m, 1H, HꢀCdO indole), δ 7.39 (m, 1H,
C4-indole), δ 6.78 (m, 1H, C7-indole), δ 6.64ꢀ6.30 (m, 3H, C2, C5,
C6-indole and 4H, phenyl), δ 4.77 (s, 2H, NꢀCH2). ESI-MS m/z 304.1
[M þ H].
1-[(4-Trifluoromethylphenyl)methyl]-1H-indole-3-carboxaldehyde
(7). Compound 7 was prepared from 1H-indole-3-carboxaldehyde
and 4-trifluoromethylbenzyl chloride as described in step A. 1H NMR
(300 MHz, DMSO-d6): δ 9.09 (m, 1H, HꢀCdO indole), δ 7.40 (m,
1H, C4-indole), δ 6.77 (m, 1H, C7-indole), δ 6.71ꢀ6.30 (m, 3H, C2,
C5, C6-indole and 4H, phenyl), δ 4.77 (s, 2H, NꢀCH2). ESI-MS m/z
304.1 [M þ H].
1-[(3-Methylphenyl)methyl]-1H-indole-3-carboxaldehyde (8).
Compound 8 was prepared from 1H-indole-3-carboxaldehyde and
1
Step E. A product from step C (1 mmol) and an isocyanate containing
C3ꢀC8 (1.1 mmol) were dissolved in anhydrous dichloromethane.
Triethylamine (1.5 mmol) and a catalytic amount of dibutyltin diacetate
were added to the solution. After it was stirred at room temperature for
30ꢀ50 min, the solvent was evaporated to cease the reaction. The
residue was dissolved in chloroform/methanol (40/1), and the product
was separated on a silica gel column. The purified product was concen-
trated, dissolved in diethyl ether, and crystallized with hexane.
Products purification was performed on silica gels or neutral alumi-
num C18 columns with a flash chromatograph system (Teledyne Tech
Inc., Thousand Oaks, CA). The purity was determined by HPLC
(Agilent Technologies 1200 Series) equipped with a C-18 bounded-
phase column (Waters, XTerra C18 MS, 3.5 μm, 4.5 mm ꢁ 50 mm).
A gradient elution was performed with acetonitrile and water as a mobile
phase and was monitored at 230 nm. All compounds used for activity
study have a purity of g95%. The chemical entities of compounds were
determined by LC/MS/UV230, 1H, and/or 13C NMR analyses. Electro-
spray ionization (ESI) MS spectra were recorded with an Agilent
LC/MSD Trap XCT Ultra spectrometer. NMR spectra were recorded
with Bruker Avance DRX 300 spectrometer.
3-methylbenzyl bromide as described in step A. H NMR (300 MHz,
CDCl3): δ 10.03 (m, 1H, HꢀCdO indole), δ 8.34 (m, 1H, C4-indole),
δ 7.73 (m, 1H, C7-indole), δ 7.39ꢀ7.14 (m, 2H, C5, C6-indole and 4H,
phenyl), δ 7.01 (m, 1H, C2-indole), δ 5.36 (s, 2H, NꢀCH2), δ 2.35 (s,
3H, ꢀCH3). ESI-MS m/z 250.0 [M þ H].
1-[(4-Methylphenyl)methyl]-1H-indole-3-carboxaldehyde (9).
Compound 9 was prepared from 1H-indole-3-carboxaldehyde and
1
4-methylbenzyl chloride as described in step A. H NMR (300 MHz,
CDCl3): δ 10.05 (m, 1H, HꢀCdO indole), δ 8.35 (m, 1H, C4-indole),
δ 7.73 (m, 1H, C7-indole), δ 7.30ꢀ6.98 (m, 3H, C2,C5, C6-indole and
4H, phenyl), δ 5.36 (s, 2H, NꢀCH2), δ 2.41(s, 3H, ꢀCH3). ESI-MS
m/z 250.0 [M þ H].
1-[(3-Nitrophenyl)methyl]-1H-indole-3-carboxaldehyde (10).
Compound 10 was prepared from 1H-indole-3-carboxaldehyde and
1
3-nitrobenzyl chloride as described in step A. H NMR (300 MHz,
CDCl3): δ 10.06 (m, 1H, HꢀCdO indole), δ 8.64 (m, 2H, phenyl), δ
8.37 (m, 1H, C4-indole), δ 7.73ꢀ7.66 (m, 1H, C7-indole, 2H, phenyl),
δ 7.32ꢀ7.10 (s, 3H, C2, C5, C6-indole), δ 5.36 (s, 2H, NꢀCH2). ESI-
MS m/z 281.0 [M þ H].
1-[(4-Nitrophenyl)methyl]-1H-indole-3-carboxaldehyde (11).
Compound 11 was prepared from 1H-indole-3-carboxaldehyde and
1-[(3-Flourophenyl)methyl]-1H-indole-3-carboxaldehyde (2).
Compound 2 was prepared from 1H-indole-3-carboxaldehyde and
1
4-nitrobenzyl chloride as described in step A. H NMR (300 MHz,
1
CDCl3): δ 10.06 (m, 1H, HꢀCdO indole), δ 8.71 (m, 2H, phenyl), δ
8.36 (m, 1H, C4-indole), δ 7.69ꢀ7.65 (m, 1H, C7-indole, 2H, phenyl),
δ 7.33ꢀ7.09 (s, 3H, C2, C5, C6-indole), δ 5.37 (s, 2H, NꢀCH2). ESI-
MS m/z 281.0 [M þ H].
3-flourobenzyl chloride as described in step A. H NMR (300 MHz,
CDCl3): δ 10.10 (m, 1H, HꢀCdO indole), δ 8.36 (m, 1H, C4-indole),
δ 7.78 (m, 1H, C7-indole), δ 7.37ꢀ7.26 (s, 2H, C5, C6-indole and 2H,
phenyl), δ 7.06ꢀ6.87 (m, 1H, C2-indole and 2H, phenyl), δ 5.40 (s, 2H,
NꢀCH2). ESI-MS m/z 254.0 [M þ H].
1-[(2,4-Dichlorophenyl)methyl]-1H-indole-3-carboxaldehyde (12).
Compound 12 was prepared from 1H-indole-3-carboxaldehyde and 2,
4-dichlorobenzyl chloride as described in step A. 1H NMR (300 MHz,
DMSO-d6): δ 9.96 (m, 1H, HꢀCdO indole), δ 8.35 (m, 1H, C4-
1-[(3-Chlorophenyl)methyl]-1H-indole-3-carboxaldehyde (3).
Compound 3 was prepared from 1H-indole-3-carboxaldehyde and
1
3-chlorobenzyl chloride as described in step A. H NMR (300 MHz,
2674
dx.doi.org/10.1021/jm101417n |J. Med. Chem. 2011, 54, 2668–2679

Journal of Medicinal Chemistry
ARTICLE
indole), δ 8.10 (m, 1H, C7-indole), δ 7.79ꢀ7.68 (s, 2H, C5, C6-indole
and 2H, phenyl), δ 7.32ꢀ7.11 (m, 1H, C2-indole and 1H, phenyl), δ
5.51 (s, 2H, NꢀCH2). ESI-MS m/z 303.9 [M þ H].
indole and 4H, phenyl), δ 7.04 (m, 1H, C2-indole), δ 5.54 (s, 2H,
NꢀCH2), δ 5.32 (s, 2H, CNꢀCH2). ESI-MS m/z 281.1 [M þ H].
1-[(2-Chlorophenyl)methyl]-1H-indole-3-ethanone (27). Com-
pound 27 was prepared from 1H-indole-3-ethanone and 2-chlorobenzyl
chloride as described in step A. δ 8.45 (m, 1H, C2-indole), δ 7.73
(m, 1H, C4-indole), δ 7.32ꢀ7.18 (s, 3H, indole and 4H, phenyl),
δ 5.35 (s, 2H, NꢀCH2), δ 2.87 (s, 3H, OdCꢀCH3). ESI-MS m/z
284.1 [M þ H].
1-[(3,4-Dibromophenyl)methyl]-1H-indole-3-carboxaldehyde (13).
Compound 13 was prepared from 1H-indole-3-carboxaldehyde and 3,4-
1
dibromobenzyl chloride as described in step A. H NMR (300 MHz,
DMSO-d6): δ9.96(m, 1H, HꢀCdO indole), δ8.49 (m, 1H, C4-indole),
δ 8.12 (m, 1H, C7-indole), δ 7.79ꢀ7.71 (s, 2H, C5, C6-indole and 1H,
phenyl), δ 7.32ꢀ7.17 (m, 1H, C2-indole and 2H, phenyl), δ 5.54 (s, 2H,
NꢀCH2). ESI-MS m/z 391.9 [M þ H].
1-[3-Chlorophenyl)methyl]-1H-indole-3-ethanone (28). Com-
pound 28 was prepared from 1H-indole-3-ethanone and 3-chlorobenzyl
chloride as described in step A. δ 8.43 (m, 1H, C2-indole), δ 7.74
(m, 1H, C4-indole), δ 7.30ꢀ7.15 (m, 3H, ꢀindole and 4H, phenyl),
δ 5.36 (s, 2H, NꢀCH2), δ 2.87 (s, 3H, OdCꢀCH3). ESI-MS m/z
284.1 [M þ H].
1-[(3-Chlorophenyl)methyl]-5-fluoro-1H-indole-3-carboxaldehyde
(16). Compound 16 was prepared from 5-fluoro-1H-indole-3-carbox-
aldehyde and 3-chlorobenzyl chloride as described in step A. 1H NMR
(300 MHz, CDCl3): δ 10.0 (m, 1H, HꢀCdO indole), δ 8.03 (m, 1H,
C7-indole), δ 7.72 (m, 1H, C4-indole), δ 7.25ꢀ7.02 (m, 2H, C2,
C6-indole and 4H, phenyl), δ 5.34 (s, 2H, NꢀCH2). ESI-MS m/z 287.9
[M þ H].
1-[4-Chlorophenyl)methyl]-1H-indole-3-ethanone (29). Com-
pound 29 was prepared from 1H-indole-3-ethanone and 4-chlorobenzyl
chloride as described in step A. δ 8.41 (m, 1H, C2-indole), δ 7.76 (m,
1H, C4-indole), δ 7.32ꢀ7.28 (m, 3H, indole, 2H, phenyl), δ 7.11ꢀ6.85
(m, 2H, phenyl), δ 5.36 (s, 2H, NꢀCH2), δ 2.56 (s, 3H, OdCꢀCH3).
13C NMR (CDCl3, 75 HMz): δ 193.1 (CdO), 136.3 (indole C-9),
134.7 (indole C-2), 134.3 (phenyl C-1), 134.1 (phenyl C-4), 129.2
(phenyl C-2 and C-6), 128.2 (phenyl C-3 and C-5), 126.4 (indole C-8),
123.6 (indole C-5), 122.8 (indole C-6), 122.7 (indole C-4), 117.7
(indole C-3), 110.0 (indole C-7), 50.1 (NꢀCH2ꢀ), 27.7 (CH3).
ESI-MS m/z 284.1 [M þ H].
1-[(4-Chlorophenyl)methyl]-5-fluoro-1H-indole-3-carboxaldehyde
(17). Compound 17 was prepared from 5-fluoro-1H-indole-3-carbox-
aldehyde and 4-chlorobenzyl chloride as described in step A. 1H NMR
(300 MHz, CDCl3): δ 10.0 (m, 1H, HꢀCdO indole), δ 8.04 (m, 1H,
C7-indole), δ 7.75 (m, 1H, C4-indole), δ 7.27ꢀ7.02 (m, 2H, C2,
C6-indole and 4H, phenyl), δ 5.36 (s, 2H, NꢀCH2). ESI-MS m/z 287.9
[M þ H].
1-[(4-Chlorophenyl)methyl]-6-fluoro-1H-indole-3-carboxaldehyde
(18). Compound 18 was prepared from 6-fluoro-1H-indole-3-carbox-
aldehyde and 4-chlorobenzyl chloride as described in step A. 1H
NMR (300 MHz, CDCl3): δ 10.0 (m, 1H, HꢀCdO indole), δ 8.29
(m, 1H, C4-indole), δ 7.85 (m, 1H, C7-indole), δ 7.36ꢀ7.27
(m, 2H, phenyl), δ 7.13ꢀ7.06 (s, 1H, C2-indole and 2H, phenyl), δ
6.95 (m, 1H, C5-indole), δ 5.30 (s, 2H, NꢀCH2). ESI-MS m/z 287.9
[M þ H].
1-(Phenylmethyl)-1H-indole-3-methanol (30). Compound 30 was
1
prepared from compound 14 as described in step C. H NMR (300
MHz, CDCl3): δ 7.65 (m, 1H, C4-indole), δ 7.32ꢀ7.03 (s, 3H, indole
and 5H, phenyl), δ 6.78 (m, 1H, C-2 indole), δ 5.27 (s, 2H, NꢀCH2), δ
4.28 (s, 2H, CH2ꢀOH). ESI-MS m/z 220.1 [M þ H ꢀ H2O].
1-(3-Fluorophenylmethyl)-1H-indole-3-methanol (31). Com-
1
pound 31 was prepared from compound 2 as described step C. H
NMR (300 MHz, CDCl3): δ 7.79 (m, 1H, C4-indole), δ 7.25ꢀ7.10
(s, 3H, indole and 1H, phenyl), δ 6.92 (m, 3H, phenyl), δ 6.81 (m, 1H,
C-2 indole), δ 5.31 (s, 2H, NꢀCH2), δ 4.91 (s, 2H, CH2ꢀOH).
ESI-MS m/z 238.1 [M þ H ꢀ H2O].
1-[(4-Chlorophenyl)methyl]-5-chloro-1H-indole-3-carboxaldehyde
(19). Compound 19 was prepared from 5-chloro-1H-indole-3-carbox-
aldehyde and 4-chlorobenzyl chloride as described in step A. 1H NMR
(300 MHz, CDCl3): δ 10.0 (m, HꢀCdO indole), δ 8.35 (m, 1H, C4-
indole), δ 7.88 (m, 1H, C7-indole), δ 7.42ꢀ7.20(m,1H, C-6 indole and
2H, phenyl), δ 7.15ꢀ7.04 (m, 1H, C2-indole and 2H, phenyl), δ 5.34
(s, 2H, NꢀCH2). ESI-MS m/z 304.0 [M þ H].
1-(4-Fluorophenylmethyl)-1H-indole-3-methanol (32). Compound
32 was prepared from 1H-indole-3-carboxaldehyde and 4-flourobenzyl
chloride as described in steps A and C. 1H NMR (300 MHz, CDCl3): δ
7.88 (m, 1H, C4-indole), δ 7.23ꢀ7.01 (s, 3H, indole and 4H, phenyl), δ
6.83 (m, 1H, C-2 indole), δ 5.27 (s, 2H, NꢀCH2), δ 4.89 (s, 2H,
CH2ꢀOH). ESI-MS m/z 238.1 [M þ H ꢀ H2O].
1-[(4-Chlorophenyl)methyl]-6-bromo-1H-indole-3-carboxalde-
hyde (20). Compound 20 was prepared from 6-bromo-1H-indole-3-
carboxaldehyde and 4-chlorobenzyl chloride as described in step A. 1H
NMR (300 MHz, CDCl3): δ 10.0 (m, HꢀCdO indole), δ 8.22 (m, 1H,
C7-indole), δ 7.92 (m, 1H, C4-indole), δ 7.47ꢀ7.32 (m, 2H, C2, C-5
indole and 2H, phenyl), δ 7.10 (m, 2H, phenyl), δ 5.32 (s, 2H,
NꢀCH2). ESI-MS m/z 349.1 [M þ H].
1-[(3-Chlorophenyl)methyl]-1H-indole-3-methanol (34). Com-
1
pound 34 was prepared from compound 16 according to step C. H
NMR (300 MHz, CDCl3): δ 7.78 (m, 1H, C4-indole), δ 7.23ꢀ7.15 (s,
3H, indole and 4H, phenyl), δ 7.00 (m, 1H, C-2 indole), δ 5.28 (s, 2H,
NꢀCH2), δ 4.91 (s, 2H, CH2ꢀOH). 13C NMR (CDCl3, 75HMz):
141.2 (phenyl C-1), 136.7 (indole C-9), 133.6 (phenyl C-3), 130.8
(phenyl C-5), 127.8 (phenyl C-2, indole C-8), 127.3 (phenyl C-6, indole
C-2), 126.3 (phenyl C-4), 121.8 (indole C-5), 119.8 (indole C-6), 119.2
(indole C-4), 116.5 (indole C-3), 110.2 (indole C-7), 55.7 (ꢀCH2OH),
48.8 (NꢀCH2). ESI-MS m/z 254.1 [M þ H ꢀ H2O].
1-[(4-Chlorophenyl)methyl]-6-chloro-2-oxoindoline-3-carboxalde-
hyde (21). Compound 21 was prepared from 6-chloro-2-oxoindole-3-
carboxaldehyde and 4-chlorobenzyl bromide as described in step A. 1H
NMR (300 MHz, DMSO-d6): δ 10.39 (m, HꢀCdO indole), δ 7.81 (m,
1H, C5-indole), δ 7.51ꢀ7.39 (m, 2H, C4, C7- indole and 2H, phenyl), δ
6.94 (m, 2H, phenyl), δ 5.40 (s, 2H, NꢀCH2, 1H, C2-indole). ESI-MS
m/z 319.5 [M þ H].
1-[(4-Chlorophenyl)methyl]-1H-indole-3-methanol (35). Com-
pound 35 was prepared from oncrasin-1 according to step C. 1H
NMR (300 MHz, CDCl3): δ 7.77 (m, 1H, C4-indole), δ 7.30ꢀ6.81
(s, 4H, indole and 4H, phenyl), δ 5.27 (s, 2H, NꢀCH2), δ 4.90 (s, 2H,
CH2ꢀOH). 13C NMR (CDCl3, 75HMz): 136.7 (indole C-9), 135.7
(phenyl C-1), 133.5 (phenyl C-4), 128.9 (phenyl C-2 and C-6), 128.2
(phenyl C-3 and C-5), 127.2 (indole C-8), 126.7 (indole C-2), 122.4
(indole C-5), 119.9 (indole C-6), 119.3 (indole C-3), 115.7 (indole
C-4), 109.7 (indole C-7), 57.2 (ꢀCH2OH), 49.4 (NꢀCH2ꢀ). ESI-MS
m/z 254.1 [M þ H ꢀ H2O].
1-[(4-Chlorophenyl)methyl]-5-fluoro-1H-indole-2-methanol (24).
Compound 24 was prepared from 5-fluoro-1H-indole-2-carboxaldehyde
and 4-chlorobenzyl chloride and then reduced as described in the step C.
1H NMR (300 MHz, CDCl3): δ 7.24 (m, 2H, phenyl), δ 7.13 (m, 1H,
C7-indole), δ 6.96ꢀ6.91 (m, 2H, C4, C6-indole, 2H, phenyl), δ 6.58
(m, 1H, C3-indole), δ 5.44 (s, 2H, NꢀCH2), δ 4.78 (s, 2H,
HOꢀCH2ꢀ). ESI-MS m/z 271.8 [M þ H ꢀ H2O].
1-[(3-Chlorophenyl)methyl]-1H-indole-3-carbonitrile (25). Com-
pound 25 was prepared from 1H-indole-3-carbonitrile and 3-chloro-
benzyl chloride as describedinstep A. 1H NMR (300 MHz, DMSO-d6): δ
8.50 (m, 1H, C4-indole), δ 7.66 (m, 2H, indole), δ 7.41ꢀ7.25 (m, 1H,
1-[(3-Bromophenyl)methyl]-1H-indole-3-methanol (36). Com-
1
pound 36 was prepared from compound 4 according to step C. H
2675
dx.doi.org/10.1021/jm101417n |J. Med. Chem. 2011, 54, 2668–2679

Journal of Medicinal Chemistry
ARTICLE
NMR (300 MHz, CDCl3): δ 7.77 (m, 1H, C4-indole), δ 7.43ꢀ7.63
(m, 2H, phenyl), δ 7.24ꢀ7.14 (s, 3H, indole, 2H, phenyl), δ 7.03
(m, 1H, C-2 indole), δ 5.27 (s, 2H, NꢀCH2), δ 4.91 (s, 2H, CH2ꢀOH).
ESI-MS m/z 315.9 [M þ H ꢀ H2O].
NMR (300 MHz, CDCl3): δ 7.75 (m, 1H, C4-indole), δ 7.50ꢀ6.88 (m,
3H, C-5,C-6, C-7-indole and 3H phenyl), δ 5.24 (s, 2H, NꢀCH2), δ
4.91 (s, 2H, CH2ꢀOH). ESI-MS m/z 375.9 [M þ H ꢀ H2O].
5-Fluoro-1-methyl-1H-indole-3-methanol (49). Compound 49 was
prepared from 5-fluoro-1- methyl-1H-indole-3-carboxaldehyde accord-
ing to step C. 1H NMR (300 MHz, CDCl3): δ 76.97ꢀ6.77 (m, 4H, C2,
C4, C6, C7-indole), δ 4.19 (s, 2H, CH2ꢀOH), δ 3.53 (s, 2H, NꢀCH3).
ESI-MS m/z 162.0 [M þ H ꢀ H2O].
1-[(4-Bromophenyl)methyl]-1H-indole-3-methanol (37). Com-
1
pound 37 was prepared from compound 5 according to step C. H
NMR (300 MHz, CDCl3): δ 7.70 (m, 1H, C4-indole), δ 7.43 (m, 2H,
phenyl), δ 7.20ꢀ7.06 (m, 3H, indole), δ 6.98ꢀ6.89 (m, 1H, C-2 indole
and 2H, phenyl), δ 5.23 (s, 2H, NꢀCH2), δ 4.26 (s, 2H, CH2ꢀOH).
ESI-MS m/z 315.9 [M þ H ꢀ H2O].
1-[(3-Fluorophenyl)methyl]-5-chloro-1H-indole-3-methanol (50).
Compound 50 was prepared from 5-chloro-1H-indole-3-carboxalde-
hyde and 3-flourobenzyl chloride according to steps A and C. 1H NMR
(300 MHz, CDCl3): δ 7.42ꢀ7.21 (m, 2H, C4, C6-indole), δ 7.16- 7.09
(m, 1H, C7-indole, 2H, phenyl), δ 6.98ꢀ6.77 (m, 1H, C-2-indole and
1-[(3-Iodiophenyl)methyl]-1H-indole-3-methanol (38). Compound
38 was prepared from 1H-indole-3-carboxaldehyde and 3-iodiobenzyl
bromide according to steps A and C. 1H NMR (300 MHz, CDCl3): δ
7.77 (m, 1H, C4-indole), δ 7.60ꢀ7.47 (m, 2H, phenyl), δ 7.20ꢀ7.04
(m, 3H,C-5,C-6, C-7-indole, 2H phenyl), δ 5.24 (s, 2H, NꢀCH2), δ
4.91 (s, 2H, CH2ꢀOH). ESI-MS m/z 346.0 [M þ H ꢀ H2O].
1-[(3-Fluoromethylphenyl)methyl]-1H-indole-3-methanol (39). Com-
pound 39 was prepared from compound 6 according to step C. 1H NMR
(300 MHz, CDCl3): δ 7.70 (m, 1H, C4-indole), δ 7.65ꢀ7.40 (m, 3H,
indole, 4H, phenyl), δ 6.91 (m, 1H, C-2 indole), δ 5.34 (s, 2H, NꢀCH2), δ
4.81 (s, 2H, CH2ꢀOH). ESI-MS m/z 288.0 [M þ H ꢀ H2O].
1-[(4-Fluoromethylphenyl)methyl]-1H-indole-3-methanol (40).
Compound 40 was prepared from compound 7 according to step C.
1H NMR (300 MHz, CDCl3): δ 7.74 (m, 1H, C4-indole), δ 7.02 (m,
2H, C-2 phenyl), δ 7.36ꢀ7.14 (m, 4H, indole, 2H, phenyl), δ 5.37 (s,
2H, NꢀCH2), δ 4.88 (s, 2H, CH2ꢀOH). ESI-MS m/z 288.0 [M þ H ꢀ
H2O].
2H, phenyl), δ 5.26 (s, 2H, NꢀCH2), δ 4.89 (s, 2H, CH2ꢀOH). 13
C
NMR (CDCl3, 75HMz): δ162.1 (phenyl C-3), 139.5 (phenyl C-1),
135.1 (indole C-9), 130.4 (phenyl C-5), 129.0 (indole C-8), 127.6
(indole C-5), 125.7 (indole C-2), 125.0 (phenyl C-6), 122.2 (indole
C-4), 119.0 (indole C-6), 115.4 (phenyl C-2), 113.6 (phenyl C-4), 113.4
(indole C-7), 112.6 (indole C-3), 57.0 (ꢀCH2OH), 49.7 (NꢀCH2ꢀ).
ESI-MS m/z 272.0 [M þ H ꢀ H2O].
1-[(4-Chlorophenyl)methyl]-5-fluoro-1H-indole-3-methanol (51).
Compound 51 was prepared from compound 17 according to step C.
1H NMR (300 MHz, CDCl3): δ 7.71 (m, 1H, C7-indole), δ 7.51 (m,
2H, phenyl), δ 7.15ꢀ6.94 (m, 3H, C-2, C-4, C-6 indole and 2H,
phenyl), δ 5.23 (s, 2H, NꢀCH2), δ 4.86 (s, 2H, CH2ꢀOH). ESI-MS m/
z 272.0 [M þ H ꢀ H2O].
1-[(4-Chlorophenyl)methyl]-6-fluoro-1H-indole-3-methanol (52).
Compound 52 was prepared from compound 18 according to step C.
1H NMR (300 MHz, CDCl3): δ 7.67 (m, 1H, C4-indole), δ 7.28 (m,
2H, phenyl), δ 7.10ꢀ7.05 (m, 2H,C-5, C-7 indole and 2H, phenyl), δ
6.85 (m, 1H, C2-indole), δ 5.27 (s, 2H, NꢀCH2), δ 4.07 (s, 2H,
CH2ꢀOH). ESI-MS m/z 272.0 [M þ H ꢀ H2O].
1-[(3-Methylphenyl)methyl]-1H-indole-3-methanol (41). Com-
pound 41 was prepared from compound 8 according to step C. 1H NMR
(300 MHz, CDCl3): δ 7.76 (m, 1H, C4-indole), δ 7.31ꢀ7.10 (s, 3H, C5,
C6, C7-indole, 4H phenyl), δ 6.97 (m, 1H, C2-indole), δ 5.24 (s, 2H,
NꢀCH2), δ 4.87 (s, 2H, CH2ꢀOH), δ 2.29 (s, 3H, PhꢀCH3). ESI-MS
m/z 233.9 [M þ H ꢀ H2O].
1-[(4-Chlorophenyl)methyl]-5-chloro-1H-indole-3-methanol (53).
Compound 53 was prepared from compound 19 according to step C.
1H NMR (300 MHz, CDCl3): δ 7.72 (m, 1H, C4-indole), δ 7.51
(m, 1H, C7-indole), δ 7.15ꢀ6.90 (m, 3H, C-2, C-4, C-5 indole and 4H,
phenyl), δ 5.23 (s, 2H, NꢀCH2), δ 4.74 (s, 2H, CH2ꢀOH). ESI-MS
m/z 288.0 [M þ H ꢀ H2O].
1-[(4-Methylphenyl)methyl]-1H-indole-3-methanol (42). Com-
pound 42 was prepared from compound 9 according to step C.
1H NMR (300 MHz, CDCl3): δ 7.75 (m, 1H, C4-indole), δ
7.32ꢀ7.06 (m, 4H, indole, 4H phenyl), δ 5.26 (s, 2H, NꢀCH2), δ
4.89 (s, 2H, CH2ꢀOH), δ 2.33 (s, 3H, PhꢀCH3). ESI-MS m/z 233.9
[M þ H ꢀ H2O].
1-[(4-Chlorophenyl)methyl]-5-methoxy-1H-indole-3-methanol
(54). Compound 54 was prepared from 5-methoxy-1H-indole-3-car-
boxaldehyde and 4-chlorobenzyl chloride according to steps A and C. 1H
NMR (300 MHz, CDCl3): δ 7.17ꢀ7.01 (m, 4H, phenyl), δ 6.95ꢀ6.72
(m, 4H, indole), δ 5.19 (s, 2H, NꢀCH2), δ 4.85 (s, 2H, CH2ꢀOH),
3.80 (s, 3H, CH3Oꢀ). ESI-MS m/z 284.1 [M þ H ꢀ H2O].
1-[(4-Methylphenyl)methyl]-5-fluoro-1H-indole-3-methanol (55).
Compound 55 was prepared from 5-fluoro-1H-indole-3-carboxaldehyde
and 4-methylbenzyl chloride according to steps A and C. 1H NMR (300
MHz, CDCl3): δ 7.28 (m, 1H, C7-indole), δ 7.18ꢀ7.02 (m, 2H, C4, C6-
indole and 4H, phenyl), δ 6.94 (m, 1H, C2-indole), δ 5.23 (s, 2H,
NꢀCH2), δ 4.83 (s, 2H, CH2ꢀOH), δ 2.19 (s, 3H, CH3ꢀPh). ESI-MS
m/z 251.8 [M þ H ꢀ H2O].
1-[(3-Nitrophenyl)methyl]-1H-indole-3-methanol (43). Compound
43 was prepared from compound 10 according to step C. 1H NMR (300
MHz, CDCl3): δ 8.14ꢀ8.06 (m, 2H, phenyl), δ 7.77 (m, 2H, phenyl), δ
7.49ꢀ7.02 (s, 5H, indole), δ 5.42 (s, 2H, NꢀCH2), δ 4.90 (s, 2H,
CH2ꢀOH). ESI-MS m/z 265.1 [M þ H ꢀ H2O].
1-[(4-Nitrophenyl)methyl]-1H-indole-3-methanol (44). Compound
44 was prepared from compound 11 according to step C. 1H NMR (300
MHz, CDCl3): δ 8.12ꢀ8.19 (m, 2H, phenyl), δ 7.79 (m, 2H, phenyl), δ
7.23ꢀ6.93 (m, 5H, indole), δ 5.42 (s, 2H, NꢀCH2), δ 4.93 (s, 2H,
CH2ꢀOH). ESI-MS m/z 265.1 [M þ H ꢀ H2O].
1-[(4-Methoxyphenyl)methyl]-1H-indole-3-methanol (45). Com-
pound 45 was prepared from 1-[(4-methoxyphenyl) methyl]-1H-in-
1
dole-3-carboxaldehyde according to step C procedure. H NMR (300
2-[1-[(4-Chlorophenyl)methyl]-1H-indole-3-]ethanol (56). Com-
pound 56 was prepared from 2-(3-hydroxyethyl) indole and 4-chlor-
obenzyl chloride as described in step A. 1H NMR (300 MHz, CDCl3): δ
7.65 (m, 1H, C4-indole), δ 7.21ꢀ7.14 (m, 3H, indole and 2H, phenyl),
δ 7.07ꢀ6.93 (m, 1H, C2-indole and 2H, phenyl), δ 5.27 (s, 2H,
NꢀCH2), δ 3.93 (s, 2H, CH2ꢀCH2ꢀOH), δ 3.06 (s, 2H,
CH2ꢀCH2ꢀOH). ESI-MS m/z 268.1 [M þ H ꢀ H2O].
MHz, CDCl3): δ 7.76 (m, 1H, C-4 indole), δ 7.34ꢀ7.29 (m, 3H, C-5,
C-6, C-7 indole and 3H, phenyl), δ 7.23ꢀ6.93 (m, 1H, C-2 indole and
1H, phenyl), δ 5.42 (s, 2H, NꢀCH2), δ 4.89 (s, 2H, CH2ꢀOH), δ 3.79
(s, 3H, CH3ꢀO). ESI-MS m/z 250.1 [M þ H ꢀ H2O].
1-[(2,4-Dichlorophenyl)methyl]-1H-indole-3-methanol (46). Com-
1
pound 46 was prepared from compound 12 according to step C. H
NMR (300 MHz, CDCl3): δ 7.80 (m, 1H, C-4 indole), δ 7.47 (m, 1H,
C-7 indole), δ 7.33ꢀ6.69 (m, 2H, C-5,C-6, indole and 3H, phenyl), δ
6.58 (m, 1H, C-2 indole), δ 5.36 (s, 2H, NꢀCH2), δ 4.92 (s, 2H,
CH2ꢀOH). ESI-MS m/z 288.0 [M þ H ꢀ H2O].
1-[1-[(4-Chlorophenyl)methyl]-1H-indole-3-]ethanol (58). Com-
1
pound 58 was prepared from compound 29 according to step C. H
NMR (300 MHz, CDCl3): δ 7.65 (m, 1H, C4-indole), δ 7.39ꢀ7.10 (m,
3H, indole and 2H, phenyl), δ 7.08ꢀ6.96 (m, 1H, C2-indole and 2H,
phenyl), δ 5.35 (s, 2H, NꢀCH2), δ 4.89 (m, 1H, CH3ꢀCHꢀOH), δ
1.47 (s, 3H, CH3ꢀCHꢀOH). ESI-MS m/z 268.1 [M þ H ꢀ H2O].
1-[(3,4-Dibromophenyl)methyl]-1H-indole-3-methanol (48). Com-
1
pound 48 was prepared from compound 13 according to step C. H
2676
dx.doi.org/10.1021/jm101417n |J. Med. Chem. 2011, 54, 2668–2679

Journal of Medicinal Chemistry
ARTICLE
1-[(4-Chlorophenyl)methyl]-1H-indole-3-methoxymethane (59).
Compound 59 was prepared from 3-methoxymethyl-1H-indole and
4-chlorobenzyl chloride as described in step A. H NMR (300 MHz,
CDCl3): δ 7.72 (m, 1H, C4-indole), δ 7.36ꢀ7.16 (m, 3H, indole and
4H, phenyl), δ 6.93 (m, 1H, C2-indole), δ 5.28 (s, 2H, NꢀCH2), 4.68
(s, 2H, ꢀCH2ꢀOCH3), 1.58 (s, 3H, ꢀCH2ꢀOCH3). ESI-MS m/z
285.9 [M þ H].
(indole 2C-6), 119.1 (indole 2C-4), 115.1 (indole 2C-3), 109.4
(indole 2C-7), 49.3 (2 NꢀCH2ꢀ), 21.2 (ꢀCH2ꢀ). ESI-MS m/z
495.0 [M þ H].
1
Bis[1-[(3-bromophenyl)methyl]-1H-indole-3-]methane (69). Com-
pound 69 was prepared from 3,30-methyleneindole and 4-bromobenzyl
bromide according to step A. 1H NMR (300 MHz, CDCl3): δ 7.65 (m,
2H, C4, C40-indole), δ 7.38 (m, 2H, C7, C70 -indole), δ 7.19ꢀ7.09 (m,
4H, C5, C6, C50, C60-indole and 4H, phenyl), δ 6.97 (m, 2H, phenyl), δ
6.83 (C2, C20-indole), δ 5.23 (s, 4H, 2 NꢀCH2), δ 4.29 (s, 2H, bridge
ꢀCH2ꢀ). ESI-MS m/z 583.0 [M þ H].
1-[(4-Chlorophenyl)methyl]-1H-indole-3-methyl Acetate (60).
Compound 60 was prepared from compound 35 and acetic anhydride
according to step D. 1H NMR (300 MHz, CDCl3): δ 7.75 (m, 1H, C4-
indole), δ 7.25ꢀ7.17(s, 3H, indole and 4H, phenyl), δ 6.92 (m, 1H, C2-
indole), δ 5.36 (s, 2H, ꢀCH2ꢀOOCH3), δ 5.29 (s, 2H, NꢀCH2), δ
2.09 (s, 3H, ꢀCH2ꢀOOCH3). ESI-MS m/z 313.9 [M þ H].
1-[(4-Chlorophenyl)methyl]-1H-indole-3-methyl Cyclohexylcarba-
mate (61). Compound 61 was prepared from compound 35 and
cyclohexyl isocyanate according to step E. 1H NMR (300 MHz, CDCl3):
δ 7.76 (m, 1H, C4-indole), δ 7.30ꢀ7.15 (s, 3H, indole and 4H, phenyl),
δ 7.05 (m, 1H, C2-indole), δ 5.33 (s, 2H, ꢀNꢀCH2), δ 5.27 (s, 2H,
indoleꢀC3ꢀCH2ꢀOꢀ), δ 3.60 (m, 1H, C1-cyclohexyl), δ 1.39ꢀ1.11
(m, 10H, cyclohexyl group), did not see ꢀNHꢀ. ESI-MS m/z 397.1
[M þ H].
Cell Culture. H460, H157, H1299, andH322 human lung cancercells
were provided by Dr. John Minna at The University of Texas South-
western Medical School (Dallas, TX). T29 immortalized normal human
ovarian epithelial cells and K-Ras-transformed T29Kt1 cells were provided
by Dr. Jinsong Liu in our institution. All cell lines were propagated
routinely in a monolayer culture in RPMI-1640 medium or in Dulbecco's
modified Eagle's medium supplemented with 10% heat-inactivated fetal
calf serum, 100 units/mL penicillin, and100μg/mL streptomycin. Allcells
were maintained in the presence of 5% CO2 at 37 °C.
Cell Viability Assay. The inhibitory effects of oncrasin-1 and other
agents on cell growth were determined by using the sulforhodamine B
assay, as described previously.8 Each experiment was performed in
quadruplicate and repeated at least three times. The IC50 value was
determined by using the CurveExpert Version 1.3 program.
1-[(4-Chlorophenyl)methyl]-1H-indole-3-carboxylic Acid (63).
1
Compound 63 was prepared from oncrasin-1 according to step B. H
NMR (300 MHz, CDCl3): δ 8.85 (s, 1H, C2-indole), δ 8.17 (m, 1H,
C4-indole), δ 7.95 (m, 1H, C7-indole), δ 7.44ꢀ7.24 (s, 2H, C5, C6-
indole and 4H, phenyl), δ 5.78 (s, 2H, NꢀCH2), did not see ꢀCOOH.
ESI-MS m/z 286.0 [M þ H].
Western Blot Analysis. Western blot analysis was performed as
we have previously described.42 Phosphorylated CTD-specific antibody
(H5, Covance, Princeton, NJ) and antibody for β-actin (Sigma) were
diluted 1:1000 before use for Western blot analysis.
Methyl
[1-[(4-Chlorophenyl)methyl]]-1H-indole-3-carboxylate
Levels of Compound 34 in Cells after Treatment. T29 and
T29Kt1 cells cultured in j15 cm plates to 80ꢀ90% confluence were
treated with 10 μM compound 34. Cells were harvested at different
time points by trypsinization. After washed once with phosphate-
buffered saline, cells were lyzed with 0.5 mL of cell lysis buffer M-PER
(Thermo Scientific). After it was homogenized in a Bullet Blender
(Next Advance Inc., Averill Park, NY) and a brief spin, the supernatant
was collected. The protein concentration was determined with BCA
protein assay reagent (Pierce, Rockford, IL). Four hundred microliters
of supernatant was mixed with 800 μL of acetonitrile to precipitate
proteins. After a brief spin at 4 °C, the clean extracts were concentrated
by nitrogen gas under pressure to 400 μL before being used for HPLC
analysis as described above to determine the levels of compound 34.
1-[(4-Chlorophenyl) methyl]-4-benzyloxy-1H-indole-3-methanol was
used as an internal standard. The levels of compound 34 were normal-
ized with protein amount in the samples.
(64). Compound 64 was prepared from methyl indole-3-carboxylate
and 4-chlorobenzyl chloride according to step A. 1H NMR (300 MHz,
CDCl3): δ 8.23 (m, 1H, C4-indole), δ 7.85 (m, 1H, C2 -indole), δ
7.35ꢀ7.29 (s, 3H, indole and 2H, phenyl), δ 7.09 (m, 2H, phenyl), 5.34
(s, 2H, NꢀCH2), δ 3.95(s, 3H, COOCH3). 13C NMR (CDCl3,
75HMz): δ 165.3 (OCdO), 136.6 (indole C-9), 134.4 (indole C-2),
134.3 (phenyl C-1), 134.0 (phenyl C-4), 129.2 (phenyl C-2 and C-6),
128.3 (phenyl C-3 and C-5), 126.8 (indole C-8), 123.1 (indole C-5),
122.1 (indole C-6), 121.9 (indole C-4), 110.1 (indole C-7), 107.8
(indole C-3), 51.0 (NꢀCH2ꢀ), 50.0 (OCH3). ESI-MS m/z 299.9
[M þ H].
1-[(4-Chlorophenyl)methyl]-1H-indole-3-methane (66). Com-
pound 66 was prepared from 3-methyl indole and 4-chlorobenzyl
chloride according to procedure of step A. 1H NMR (300 MHz,
CDCl3): δ 7.61 (m, 1H, C4-indole), δ 7.20ꢀ7.02 (m, 3H, indole and
4H, phenyl), δ 6.90 (m, 1H, C2-indole), 5.25 (s, 2H, NꢀCH2), δ 2.35
(s, 3H, indole-3-CH3). ESI-MS m/z 256.0 [M þ H].
’ AUTHOR INFORMATION
Methyl 2-[1-[(4-Chlorophenyl)methyl]-1H-indole-3-methylami-
no]acetate (67). Compound 67 was prepared from 1-[(4-chloro-
phenyl) methyl]-1H-indole-3-carboxaldehyde and glycinmethylester
Corresponding Author
*Tel: 713-563-9147. Fax: 713-794-4901. E-mail: bfang@
mdanderson.org.
1
hydrochloride according to step A. H NMR (300 MHz, CDCl3): δ
7.73 (m, 2H, C4, C7-indole), δ 7.22ꢀ7.02 (m, 2H, indole and 4H,
phenyl), δ 6.90 (m, 1H, C2-indole), 5.25 (s, 2H, NꢀCH2), δ 4.01 (s,
2H, CH2ꢀNHꢀCH2ꢀCOOCH3), δ 3.73 (s, 3H, CH2ꢀNHꢀ
CH2ꢀCOOCH3), δ 3.51 (s, 2H, CH2ꢀNHꢀCH2ꢀCOOCH3). ESI-
MS m/z 341.8 [M þ H ꢀ H].
’ ACKNOWLEDGMENT
We thank Dr. Joel Morris at the National Institute of Health
for helpful suggestions and Stephanie Deming for editorial
review of this manuscript. This work was supported in part by
Cancer Center Support Grant CA-016672 to The University of
Texas MD Anderson Cancer Center, which supports the Lung
Program, the Nuclear Magnetic Resonance Facility, and the
DNA Analysis Facility; National Cancer Institute grants R01
CA 092487 and R01 CA 124951 (B.F.); the Rapid Access to
Interventional Development Program; INNOFUND of China
(09C26213301228); and National Natural Science Foundation
of China No. 30973563 (XW.).
Bis[1-[(3-chlorophenyl)methyl]-1H-indole-3-]methane (68). Com-
pound 68 was prepared from 3,30-methyleneindole and 4-chlorobenzyl
chloride according to step A. 1H NMR (300 MHz, CDCl3): δ 7.65 (m,
2H, C4, C40--indole), δ 7.23ꢀ7.17 (s, 6H, C5, C6, C7, C50, C60, C70-
indole), δ 7.15ꢀ7.10 (m, 6H, phenyl), δ 6.93ꢀ6.34 (m, 2H, C2, C20-
indole, 2H phenyl), 5.29 (s, 4H, 2 NꢀCH2), δ 4.29 (s, 2H, bridge
ꢀCH2ꢀ). 13C NMR (CDCl3, 75HMz): δ 140.0 (indole 2C-9), 136.7
(phenyl 2C-1), 134.6 (phenyl 2C-3), 130.0 (phenyl 2C-5), 128.3
(phenyl 2C-2), 127.7 (indole 2C-8), 126.7 (phenyl 2C-6), 126.3
(indole 2C-2), 124.7 (phenyl 2C-4), 121.9 (indole 2C-5), 119.5
2677
dx.doi.org/10.1021/jm101417n |J. Med. Chem. 2011, 54, 2668–2679

Journal of Medicinal Chemistry
ARTICLE
’ ABBREVIATIONS USED
(9) Kaelin, W. G., Jr. The concept of synthetic lethality in the context
of anticancer therapy. Nature Rev. Cancer 2005, 5, 689–698.
(10) Bryant, H. E.; Schultz, N.; Thomas, H. D.; Parker, K. M.;
Flower, D.; Lopez, E.; Kyle, S.; Meuth, M.; Curtin, N. J.; Helleday, T.
Specific killing of BRCA2-deficient tumours with inhibitors of poly-
(ADP-ribose) polymerase. Nature 2005, 434, 913–917; Erratum
appears in Nature 2007, 447 (7142), 346.
CDK, cyclin-dependent kinase; CTD, C-terminal domain; HPLC,
high-performance liquid chromatography; IC50, dose that causes a
50% reduction in the number of surviving cells as compared with the
number of surviving cells in the control group; MS, mass spectro-
metry;NMR, nuclear magnetic resonance
(11) Farmer, H.; McCabe, N.; Lord, C. J.; Tutt, A. N.; Johnson,
D. A.; Richardson, T. B.; Santarosa, M.; Dillon, K. J.; Hickson, I.;
Knights, C.; Martin, N. M.; Jackson, S. P.; Smith, G. C.; Ashworth, A.
Targeting the DNA repair defect in BRCA mutant cells as a therapeutic
strategy. Nature 2005, 434, 917–921.
(12) Koumenis, C.; Giaccia, A. Transformed cells require contin-
uous activity of RNA polymerase II to resist oncogene-induced apop-
tosis. Mol. Cell. Biol. 1997, 17, 7306–7316.
(13) Baumli, S.; Lolli, G.; Lowe, E. D.; Troiani, S.; Rusconi, L.;
Bullock, A. N.; Debreczeni, J. E.; Knapp, S.; Johnson, L. N. The
structure of P-TEFb (CDK9/cyclin T1), its complex with flavopiridol
and regulation by phosphorylation. EMBO J. 2008, 27, 1907–1918.
(14) Chao, S. H.; Price, D. H. Flavopiridol inactivates P-TEFb and
blocks most RNA polymerase II transcription in vivo. J. Biol. Chem.
2001, 276, 31793–31799.
(15) Maccallum, D. E.; Melville, J.; Frame, S.; Watt, K.; Anderson, S.;
Gianella-Borradori, A.; Lane, D. P.; Green, S. R. Seliciclib (CYC202,
R-Roscovitine) induces cell death in multiple myeloma cells by inhibi-
tion of RNA polymerase II-dependent transcription and down-regula-
tion of Mcl-1. Cancer Res. 2005, 65, 5399–5407.
’ REFERENCES
(1) Ding, L.; Getz, G.; Wheeler, D. A.; Mardis, E. R.; McLellan,
M. D.; Cibulskis, K.; Sougnez, C.; Greulich, H.; Muzny, D. M.; Morgan,
M. B.; Fulton, L.; Fulton, R. S.; Zhang, Q.; Wendl, M. C.; Lawrence,
M. S.; Larson, D. E.; Chen, K.; Dooling, D. J.; Sabo, A.; Hawes, A. C.;
Shen, H.; Jhangiani, S. N.; Lewis, L. R.; Hall, O.; Zhu, Y.; Mathew, T.;
Ren, Y.; Yao, J.; Scherer, S. E.; Clerc, K.; Metcalf, G. A.; Ng, B.;
Milosavljevic, A.; Gonzalez-Garay, M. L.; Osborne, J. R.; Meyer, R.;
Shi, X.; Tang, Y.; Koboldt, D. C.; Lin, L.; Abbott, R.; Miner, T. L.; Pohl,
C.; Fewell, G.; Haipek, C.; Schmidt, H.; Dunford-Shore, B. H.; Kraja, A.;
Crosby, S. D.; Sawyer, C. S.; Vickery, T.; Sander, S.; Robinson, J.;
Winckler, W.; Baldwin, J.; Chirieac, L. R.; Dutt, A.; Fennell, T.; Hanna,
M.; Johnson, B. E.; Onofrio, R. C.; Thomas, R. K.; Tonon, G.; Weir,
B. A.; Zhao, X.; Ziaugra, L.; Zody, M. C.; Giordano, T.; Orringer, M. B.;
Roth, J. A.; Spitz, M. R.; Wistuba, I. I.; Ozenberger, B.; Good, P. J.;
Chang, A. C.; Beer, D. G.; Watson, M. A.; Ladanyi, M.; Broderick, S.;
Yoshizawa, A.; Travis, W. D.; Pao, W.; Province, M. A.; Weinstock,
G. M.; Varmus, H. E.; Gabriel, S. B.; Lander, E. S.; Gibbs, R. A.;
Meyerson, M.; Wilson, R. K. Somatic mutations affect key pathways in
lung adenocarcinoma. Nature 2008, 455, 1069–1075.
(16) Chen, R.; Keating, M. J.; Gandhi, V.; Plunkett, W. Transcription
inhibition by flavopiridol: mechanism of chronic lymphocytic leukemia
cell death. Blood 2005, 106, 2513–2519.
(17) Wittmann, S.; Bali, P.; Donapaty, S.; Nimmanapalli, R.; Guo, F.;
Yamaguchi, H.; Huang, M.; Jove, R.; Wang, H. G.; Bhalla, K. Flavopiridol
down-regulates antiapoptotic proteins and sensitizes human breast cancer
cells to epothilone B-induced apoptosis. Cancer Res. 2003, 63, 93–99.
(18) Raje, N.; Kumar, S.; Hideshima, T.; Roccaro, A.; Ishitsuka, K.;
Yasui, H.; Shiraishi, N.; Chauhan, D.; Munshi, N. C.; Green, S. R.;
Anderson, K. C. Seliciclib (CYC202 or R-roscovitine), a small-molecule
cyclin-dependent kinase inhibitor, mediates activity via down-regulation
of Mcl-1 in multiple myeloma. Blood 2005, 106, 1042–1047.
(19) Guo, W.; Wu, S.; Wang, L.; Wang, R.; Wei, L.; Liu, J.; Fang, B.
Interruption of RNA processing machinery by a small compound
1-[(4-chlorophenyl) methyl]-1H-indole-3-carboxaldehyde (oncrasin-1).
Mol. Cancer Ther. 2009, 8, 441–448.
(20) Archambault, J.; Chambers, R. S.; Kobor, M. S.; Ho, Y.; Cartier,
M.; Bolotin, D.; Andrews, B.; Kane, C. M.; Greenblatt, J. An essential
component of a C-terminal domain phosphatase that interacts with
transcription factor IIF in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci.
U.S.A. 1997, 94, 14300–14305.
(21) Bird, G.; Zorio, D. A.; Bentley, D. L. RNA polymerase II
carboxy-terminal domain phosphorylation is required for cotranscrip-
tional pre-mRNA splicing and 30-end formation. Mol. Cell. Biol. 2004,
24, 8963–8969.
(2) Wood, L. D.; Parsons, D. W.; Jones, S.; Lin, J.; Sjoblom, T.;
Leary, R. J.; Shen, D.; Boca, S. M.; Barber, T.; Ptak, J.; Silliman, N.;
Szabo, S.; Dezso, Z.; Ustyanksky, V.; Nikolskaya, T.; Nikolsky, Y.;
Karchin, R.; Wilson, P. A.; Kaminker, J. S.; Zhang, Z.; Croshaw, R.;
Willis, J.; Dawson, D.; Shipitsin, M.; Willson, J. K.; Sukumar, S.; Polyak,
K.; Park, B. H.; Pethiyagoda, C. L.; Pant, P. V.; Ballinger, D. G.; Sparks,
A. B.; Hartigan, J.; Smith, D. R.; Suh, E.; Papadopoulos, N.; Buckhaults,
P.; Markowitz, S. D.; Parmigiani, G.; Kinzler, K. W.; Velculescu, V. E.;
Vogelstein, B. The genomic landscapes of human breast and colorectal
cancers. Science 2007, 318, 1108–1113.
(3) Lynch, T. J.; Bell, D. W.; Sordella, R.; Gurubhagavatula, S.;
Okimoto, R. A.; Brannigan, B. W.; Harris, P. L.; Haserlat, S. M.; Supko,
J. G.; Haluska, F. G.; Louis, D. N.; Christiani, D. C.; Settleman, J.; Haber,
D. A. Activating mutations in the epidermal growth factor receptor
underlying responsiveness of non-small-cell lung cancer to gefitinib.
N. Engl. J. Med. 2004, 350, 2129–2139.
(4) Druker, B. J.; Tamura, S.; Buchdunger, E.; Ohno, S.; Segal, G. M.;
Fanning, S.; Zimmermann, J.; Lydon, N. B. Effects of a selective inhibitor
of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nature
Med. 1996, 2, 561–566.
(5) Wilhelm, S. M.; Carter, C.; Tang, L.; Wilkie, D.; McNabola, A.;
Rong, H.; Chen, C.; Zhang, X.; Vincent, P.; McHugh, M.; Cao, Y.;
Shujath, J.; Gawlak, S.; Eveleigh, D.; Rowley, B.; Liu, L.; Adnane, L.;
Lynch, M.; Auclair, D.; Taylor, I.; Gedrich, R.; Voznesensky, A.; Riedl,
B.; Post, L. E.; Bollag, G.; Trail, P. A. BAY 43-9006 exhibits broad
spectrum oral antitumor activity and targets the RAF/MEK/ERK
pathway and receptor tyrosine kinases involved in tumor progression
and angiogenesis. Cancer Res. 2004, 64, 7099–7109.
(22) Hirose, Y.; Tacke, R.; Manley, J. L. Phosphorylated RNA
polymerase II stimulates pre-mRNA splicing. Genes Dev. 1999,
13, 1234–1239.
(23) Kim, E.; Du, L.; Bregman, D. B.; Warren, S. L. Splicing factors
associate with hyperphosphorylated RNA polymerase II in the absence
of pre-mRNA. J. Cell Biol. 1997, 136, 19–28.
(24) McCracken, S.; Fong, N.; Yankulov, K.; Ballantyne, S.; Pan, G.;
Greenblatt, J.; Patterson, S. D.; Wickens, M.; Bentley, D. L. The
C-terminal domain of RNA polymerase II couples mRNA processing
to transcription. Nature 1997, 385, 357–361.
(25) Millhouse, S.; Manley, J. L. The C-terminal domain of RNA
polymerase II functions as a phosphorylation-dependent splicing acti-
vator in a heterologous protein. Mol. Cell. Biol. 2005, 25, 533–544.
(26) Misteli, T.; Spector, D. L. RNA polymerase II targets pre-
mRNA splicing factors to transcription sites in vivo. Mol. Cell 1999,
3, 697–705.
(6) Torrance, C. J.; Agrawal, V.; Vogelstein, B.; Kinzler, K. W. Use of
isogenic human cancer cells for high-throughput screening and drug
discovery. Nat. Biotechnol. 2001, 19, 940–945.
(7) Dolma, S.; Lessnick, S. L.; Hahn, W. C.; Stockwell, B. R.
Identification of genotype-selective antitumor agents using synthetic
lethal chemical screening in engineered human tumor cells. Cancer Cell
2003, 3, 285–296.
(8) Guo, W.; Wu, S.; Liu, J.; Fang, B. Identification of a small
molecule with synthetic lethality for K-ras and protein kinase C iota.
Cancer Res. 2008, 68, 7403–7408.
2678
dx.doi.org/10.1021/jm101417n |J. Med. Chem. 2011, 54, 2668–2679

Journal of Medicinal Chemistry
ARTICLE
(27) Chinni, S. R.; Li, Y.; Upadhyay, S.; Koppolu, P. K.; Sarkar, F. H.
Indole-3-carbinol (I3C) induced cell growth inhibition, G1 cell cycle
arrest and apoptosis in prostate cancer cells. Oncogene 2001,
20, 2927–2936.
(28) Garcia, H. H.; Brar, G. A.; Nguyen, D. H.; Bjeldanes, L. F.;
Firestone, G. L. Indole-3-carbinol (I3C) inhibits cyclin-dependent
kinase-2 function in human breast cancer cells by regulating the size
distribution, associated cyclin E forms, and subcellular localization of the
CDK2 protein complex. J. Biol. Chem. 2005, 280, 8756–8764.
(29) Liu, J.; Yang, G.; Thompson-Lanza, J. A.; Glassman, A.; Hayes,
K.; Patterson, A.; Marquez, R. T.; Auersperg, N.; Yu, Y.; Hahn, W. C.;
Mills, G. B.; Bast, R. C., Jr. A genetically defined model for human
ovarian cancer. Cancer Res. 2004, 64, 1655–1663.
(30) Anderton, M. J.; Manson, M. M.; Verschoyle, R. D.; Gescher,
A.; Lamb, J. H.; Farmer, P. B.; Steward, W. P.; Williams, M. L.
Pharmacokinetics and tissue disposition of indole-3-carbinol and its
acid condensation products after oral administration to mice. Clin.
Cancer Res. 2004, 10, 5233–5241.
(31) Oelgeschlager, T. Regulation of RNA polymerase II activity by
CTD phosphorylation and cell cycle control. J. Cell. Physiol. 2002,
190, 160–169.
(32) Mortillaro, M. J.; Blencowe, B. J.; Wei, X.; Nakayasu, H.; Du, L.;
Warren, S. L.; Sharp, P. A.; Berezney, R. A hyperphosphorylated form of
the large subunit of RNA polymerase II is associated with splicing
complexes and the nuclear matrix. Proc. Natl. Acad. Sci. U.S.A. 1996,
93, 8253–8257.
(33) Larochelle, S.; Chen, J.; Knights, R.; Pandur, J.; Morcillo, P.;
Erdjument-Bromage, H.; Tempst, P.; Suter, B.; Fisher, R. P. T-loop
phosphorylation stabilizes the CDK7-cyclin H-MAT1 complex in vivo
and regulates its CTD kinase activity. EMBO J. 2001, 20, 3749–3759.
(34) Shiekhattar, R.; Mermelstein, F.; Fisher, R. P.; Drapkin, R.;
Dynlacht, B.; Wessling, H. C.; Morgan, D. O.; Reinberg, D. Cdk-
activating kinase complex is a component of human transcription factor
TFIIH. Nature 1995, 374, 283–287.
(35) Akoulitchev, S.; Chuikov, S.; Reinberg, D. TFIIH is negatively
regulated by cdk8-containing mediator complexes. Nature 2000,
407, 102–106.
(36) Leclerc, V.; Tassan, J. P.; O'Farrell, P. H.; Nigg, E. A.; Leopold,
P. Drosophila Cdk8, a kinase partner of cyclin C that interacts with the
large subunit of RNA polymerase II. Mol. Biol. Cell 1996, 7, 505–513.
(37) Kim, Y. K.; Bourgeois, C. F.; Isel, C.; Churcher, M. J.; Karn, J.
Phosphorylation of the RNA polymerase II carboxyl-terminal domain by
CDK9 is directly responsible for human immunodeficiency virus type 1
Tat-activated transcriptional elongation. Mol. Cell. Biol. 2002, 22, 4622–
4637.
(38) Peterson, S. R.; Jesch, S. A.; Chamberlin, T. N.; Dvir, A.;
Rabindran, S. K.; Wu, C.; Dynan, W. S. Stimulation of the DNA-
dependent protein kinase by RNA polymerase II transcriptional acti-
vator proteins. J. Biol. Chem. 1995, 270, 1449–1454.
(39) Bonnet, F.; Vigneron, M.; Bensaude, O.; Dubois, M. F. Tran-
scription-independent phosphorylation of the RNA polymerase II
C-terminal domain (CTD) involves ERK kinases (MEK1/2). Nucleic
Acids Res. 1999, 27, 4399–4404.
(40) Baskaran, R.; Chiang, G. G.; Wang, J. Y. Identification of a
binding site in c-Ab1 tyrosine kinase for the C-terminal repeated domain
of RNA polymerase II. Mol. Cell. Biol. 1996, 16, 3361–3369.
(41) Baskaran, R.; Dahmus, M. E.; Wang, J. Y. Tyrosine phosphor-
ylation of mammalian RNA polymerase II carboxyl-terminal domain.
Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 11167–11171.
(42) Wu, S.; Zhu, H.; Gu, J.; Zhang, L.; Teraishi, F.; Davis, J. J.;
Jacob, D. A.; Fang, B. Induction of Apoptosis and Down-regulation
of Bcl-XL in Cancer Cells by a novel small molecule 2[[3-(2,3-
dichlorophenoxy)propyl]amino] ethanol (2,3-DCPE). Cancer Res.
2003, 64, 1110–1113.
2679
dx.doi.org/10.1021/jm101417n |J. Med. Chem. 2011, 54, 2668–2679