Synthesis and identification of small molecules that potently induce apoptosis in melanoma cells through G1 cell cycle arrest

Article abstract of DOI:10.1021/ja042913p

Late-stage malignant melanoma is a cancer that is refractory to current chemotherapeutic treatments. The average survival time for patients with such a diagnosis is 6 months. In general, the vast majority of anticancer drugs operate through induction of cell cycle arrest and cell death in either the DNA synthesis (S) or mitosis (M) phase of the cell cycle. Unfortunately, the same mechanisms that melanocytes possess to protect cells from DNA damage often confer resistance to drugs that derive their toxicity from S or M phase arrest. Described herein is the synthesis of a combinatorial library of potential proapoptotic agents and the subsequent identification of a class of small molecules (triphenylmethylamides, TPMAs) that arrest the growth of melanoma cells in the G1 phase of the cell cycle. Several of these TPMAs are quite potent inducers of apoptotic death in melanoma cell lines (IC₅₀ ～ 0.5 μM), and importantly, some TPMAs are comparatively nontoxic to normal cells isolated from the bone marrow of healthy donors. Furthermore, the TPMAs were found to dramatically reduce the level of active nuclear factor κ-B (NFκB) in the cell; NFκB is known to be constitutively active in melanoma, and this activity is critical for the proliferation of melanoma cells and their evasion of apoptosis. Compounds that reduce the level of NFκB and arrest cells in the G1 phase of the cell cycle can provide insights into the biology of melanoma and may be effective antimelanoma agents.

Full text of DOI:10.1021/ja042913p

Published on Web 05/26/2005
Synthesis and Identification of Small Molecules that Potently
Induce Apoptosis in Melanoma Cells through G1 Cell
Cycle Arrest
Robin S. Dothager,† Karson S. Putt,† Brittany J. Allen,‡ Benjamin J. Leslie,‡
Vitaliy Nesterenko,‡ and Paul J. Hergenrother*,†,‡
Contribution from the Department of Chemistry and Department of Biochemistry,
Roger Adams Laboratory, UniVersity of Illinois, Urbana, Illinois 61801
Received November 23, 2004; E-mail: hergenro@uiuc.edu
Abstract: Late-stage malignant melanoma is a cancer that is refractory to current chemotherapeutic
treatments. The average survival time for patients with such a diagnosis is 6 months. In general, the vast
majority of anticancer drugs operate through induction of cell cycle arrest and cell death in either the DNA
synthesis (S) or mitosis (M) phase of the cell cycle. Unfortunately, the same mechanisms that melanocytes
possess to protect cells from DNA damage often confer resistance to drugs that derive their toxicity from
S or M phase arrest. Described herein is the synthesis of a combinatorial library of potential proapoptotic
agents and the subsequent identification of a class of small molecules (triphenylmethylamides, TPMAs)
that arrest the growth of melanoma cells in the G1 phase of the cell cycle. Several of these TPMAs are
quite potent inducers of apoptotic death in melanoma cell lines (IC50 ∼ 0.5 µM), and importantly, some
TPMAs are comparatively nontoxic to normal cells isolated from the bone marrow of healthy donors.
Furthermore, the TPMAs were found to dramatically reduce the level of active nuclear factor κ-B (NFκB)
in the cell; NFκB is known to be constitutively active in melanoma, and this activity is critical for the
proliferation of melanoma cells and their evasion of apoptosis. Compounds that reduce the level of NFκB
and arrest cells in the G1 phase of the cell cycle can provide insights into the biology of melanoma and
may be effective antimelanoma agents.
Introduction
while etoposide, taxol, and colchicine all target operations that
affect mitosis and ultimately result in M phase arrest.8,9
A common trait of most cancers is the ability to evade the
natural cell death process.1 Although healthy cells from higher
eukaryotes have tightly regulated mechanisms for apoptosis, or
programmed cell death, cancerous cells often have multiple
means by which they evade this pathway and achieve immortal-
ity. Virtually every point on the apoptotic cascade has been
exploited by cancer, typically through aberrant expression levels
or inactivating mutations in certain key proteins.2-4 A goal of
many anticancer treatments is to overcome this endogenous
resistance to apoptosis, and indeed, the majority of anticancer
drugs function by inducing apoptotic cell death.
Currently, 75% of cancer deaths are due to epithelial
cancers;10 some of these forms of cancer, such as advanced
malignant melanoma, are largely incurable. The five-year
survival rates for patients with disseminated melanoma is <5%11
with an average survival time of 6-10 months.12 The lack of
sensitivity of melanoma to chemotherapy has been well-
documented.13,14 Common anticancer drugs, such as taxol,
cisplatin, etoposide, doxorubicin, and several others, have shown
no efficacy in large randomized trials,15 and even popular
combination therapies have provided no benefits to melanoma
patients.16 This lack of clinical efficacy is supported by in vitro
studies testing drugs against melanoma cell lines.17 In fact, only
The cell cycle is divided into four phases: G1, S (DNA
synthesis), G2, and M (mitosis). Anticancer agents typically
target the propensity of cancer cells to rapidly replicate their
DNA and divide and thus arrest cell growth in the S or M phase
of the cell cycle. For instance, cisplatin, doxorubicin, and
cyclophosphamide cause DNA damage and S phase arrest,5-7
(6) Minotti, G.; Menna, P.; Salvatorelli, E.; Cairo, G.; Gianni, L. Pharmacol.
ReV. 2004, 56, 185-229.
(7) Schwartz, P. S.; Waxman, D. J. Mol. Pharmacol. 2001, 60, 1268-1279.
(8) Clifford, B.; Beljin, M.; Stark, G. R.; Taylor, W. R. Cancer Res. 2003, 63,
4074-4081.
(9) Jordan, M. A.; Wilson, L. Nature ReV. Cancer 2004, 4, 253.
(10) Adjei, A. A.; Rowinsky, E. K. Cancer Biol. Ther. 2003, S1, S5-S15.
(11) McGovern, V. J.; Balch, C. M.; Milton, G. W. In Cutaneous Melanoma:
Clinical Management and Treatment Results Worldwide; Lippincott,
Williams & Wilkins: Philadelphia, 1985; pp 29-42.
(12) Jemal, A.; Thomas, A.; Murray, T.; Thun, M. CA Cancer J. Clin. 2002,
52, 23-47.
† Department of Biochemistry.
‡ Department of Chemistry.
(1) Hanahan, D.; Weinberg, R. A. Cell 2000, 100, 57-70.
(2) Makin, G.; Dive, C. Trends Mol. Med. 2003, 9, 251-255.
(3) Ehlert, J. E.; Kubbutat, M. H. Onkologie 2001, 24, 433-440.
(4) Zornig, M.; Hueber, A.; Baum, W.; Evan, G. Biochim. Biophys. Acta 2001,
1551, F1-F37.
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2001, 93, 617-622.
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2000, 19, 21-34.
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J. AM. CHEM. SOC. 2005, 127, 8686-8696
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Arrest of Melanoma Cell Growth in the G1 Phase
A R T I C L E S
one single-entity drug, dacarbazine (DTIC), has been approved
by the FDA for treatment of late-stage melanoma, and this
medicine provides complete remission in only 2% of pa-
tients.14,18 Thus, as common anticancer drugs are profoundly
ineffective in the treatment of disseminated melanoma, there is
a clear need for compounds that are selectively cytotoxic to
cancerous melanocytes and that act through nonstandard anti-
cancer mechanisms. Some urgency is required in this regard,
as the lifetime risk for melanoma is increasing and now
estimated at 1 in 75, and it is a disease that is as likely to afflict
the young as the old.12
The poor long-term prognosis for late-stage melanoma
patients is due to the intrinsic resistance of malignant melano-
cytes to mechanisms of apoptotic death as induced by common
anticancer drugs. In their natural role as a protectant from the
harmful effects of the sun, melanocytes are bombarded with
UV light, a potent DNA damaging agent. Therefore, it is not
surprising that melanoma cells are exquisitely resistant to
therapies that target DNA synthesis and replication through
radiation and DNA alkylation; indeed, some lines of evidence
indicate that such treatments cause melanoma cells to prolifer-
ate.19 Recently, examination of apoptotic and cell checkpoint
proteins has revealed clues to how melanoma cells evade cell
death and continue to proliferate. It is now known that certain
melanomas have a methylation-inactivated Apaf-1 signaling
complex,20 and/or exhibit upregulation of the caspase-inhibiting
protein survivin.21 In addition, a common chromosomal defect
in melanoma cells is a deletion of the INK4a/ARF locus in the
9p21 region, resulting in the inactivation of the G1/S checkpoint
through ablation of the p16INK4A protein.22,23 A final trait of
cancerous melanocytes is the constitutive upregulation of active
nuclear factor κ-B (NFκB). This increased level of active NFκB
is thought to contribute to the resistance of melanoma to
apoptosis-inducing chemotherapeutic agents.24,25
Numerous trials have demonstrated that melanoma cells are
resistant to standard anticancer drugs. The goal of this study
was to identify compounds that are highly potent against cancer
cells in culture and that act through a mechanism that arrests
cell development in the G1 phase of the cell cycle. We
hypothesized that such compounds might be effective against
melanoma because, unlike S phase arresting compounds,
melanoma cells should have no intrinsic resistance to G1 phase
arrestors. In addition, a compound that arrests growth in the
G1 phase would prevent the cell from passing through the
critical G1/S checkpoint that is disrupted via mutation in
melanoma cells and could have a novel macromolecular target.
Herein we report the synthesis and screening of a library of
100 potential apoptotic inducers and the identification of
compounds that arrest cell growth in the G1 phase of the cell
cycle and induce apoptosis in melanoma cells. While the hits
from this first library were quite toxic to melanoma cells, they
were also found to be toxic to noncancerous cells from human
bone marrow. Therefore, a second-generation library of 104
compounds was created and screened. Several of these com-
pounds have powerful proapoptotic activity in melanoma cell
lines and G1 phase arresting capacity. In addition, some of these
compounds are markedly less toxic to noncancerous control
cells. These compounds reduce the amount of active NFκB in
the cell, which is known to be constitutively active in melanoma
and directly linked to proliferation and apoptosis inhibition.
Given the poor long-term survival prospects for patients with
late-stage melanoma and the lack of any effective treatments,
compounds discovered through this strategy can provide im-
portant insights into the biology of melanoma and have potential
as chemotherapeutic agents.
Results and Discussion
Synthesis and Screening of the First-Generation Library.
To create compounds that might induce apoptosis, a small
molecule library was designed based on the known apoptosis-
inducing properties of certain amides.26-28 The building blocks
used to construct this library are depicted in Scheme 1. To form
the amide bond, carboxylic acids 1-10 were first converted to
the acid chlorides and then treated in parallel with amines A-J
(1.05 equiv) and triethylamine (1.05 equiv). All reactions were
performed on a 0.05 mmol scale such that approximately 20
mg of pure product would be obtained (assuming 100% yield
for a compound of 400 MW). After the amidation reaction, all
compounds were purified through small plugs of silica gel. This
protocol provided a high yield (average yield ) 94%) of highly
pure compounds; all 100 compounds were assessed by HPLC-
MS, and this library had an average purity of 89% (see
Supporting Information for the exact purity of individual
compounds). The precise milligram amount produced was
determined for every compound, enabling each sample to be
prepared at an exact and identical micromolar concentration for
high-throughput biological testing.
The ability of the 100 amides thus produced to induce death
in cancer cells was assessed by an assay based on the reduction
of the dye MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-
methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt].
This tetrazolium dye is rapidly converted to the formazan
product by living cells, and therefore provides a sensitive readout
of cell life or death that can be monitored spectroscopically.
As shown in Figure 1, several of the compounds rapidly (within
24 h) and potently induced death at 50 µM in the U-937 cell
line, an easy-to-culture lymphoma cell line used during this
initial screening stage. Data on the dose dependence of death-
inducing activity was gathered for the most active compounds
as identified through this high-throughput screen. As shown in
Table 1, multiple compounds induced cell death with IC50 values
in the low micromolar range.
(16) Middleton, M. R.; Lorigan, P.; Owen, J.; Ashcroft, L.; Lee, S. M.; Harper,
P.; Thatcher, N. Br. J. Cancer 2000, 82, 1158-1162.
(17) Schadendorf, D.; Worm, M.; Algermissen, B.; Kohlmus, C. M.; Czarnetzki,
B. M. Cancer 1994, 73, 103-108.
(18) Anderson, C. M.; Buzaid, A. C.; Legha, S. S. Oncology 1995, 9, 1149-
1154.
Cell Cycle Analysis. The data in Figure 1 and Table 1 show
obvious structure-activity trends. Certain amides derived from
the triphenylpropionic acid 1 and triphenylacetic acid 4 building
blocks induce a high degree of cell death in this assay, and
(19) Lev, D. C.; et al. J. Clin. Oncol. 2004, 22, 2092-2100.
(20) Soengas, M. S.; et al. Nature 2001, 409, 207-211.
(21) Grossman, D.; McNiff, J. M.; Li, F.; Altieri, D. C. J. InVest. Dermatol.
1999, 113, 1076-1081.
(22) Cannon-Albright, L. A.; et al. Science 1992, 258, 1148-1152.
(23) Fountain, J. W.; et al. Proc. Natl. Acad. Sci. 1992, 89, 10557-10561.
(24) Huang, S.; DeGuzman, A.; Bucana, C. D.; Fidler, I. J. Clin. Cancer Res.
2000, 6, 2573-2581.
(26) Nesterenko, V.; Putt, K. S.; Hergenrother, P. J. J. Am. Chem. Soc. 2003,
125, 14672-14673.
(27) Nguyen, J. T.; Wells, J. A. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 7533-
7538.
(25) Makarov, S. S. Mol. Med. Today 2000, 6, 441-448.
(28) Hwang, S. Y.; et al. Int. J. Oncol. 2003, 22, 151-157.
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Scheme 1. One Hundred Amides Were Synthesized in Parallel and Then Filtered through Small Plugs of Silica Gela
a The compounds had an average purity of 89% as assessed by LC-MS.
Figure 1. Percent of cell death induced in U-937 cells by the amide products depicted in Scheme 1. Cell death was evaluated after a 24-h incubation with
compound at a concentration of 50 µM; death was quantitated by a MTS-bioreduction assay.
several other compounds have IC50 values in the low micromolar
range. At this point, experiments were conducted to determine
the phase of the cell cycle in which these compounds arrest
cell growth. This is typically determined by assessing the DNA
content of cells stained with propidium iodide as measured by
flow cytometry. Flow cytometry data of U-937 cells treated with
selected compounds are presented in Figure 2, along with control
data. As expected, controls with compounds known to induce
M phase arrest such as the microtubule stabilizer taxol (Figure
2A) show a pronounced shift to cell populations with 4n DNA,
indicative of G2/M phase arrest. One of the active cell death
inducers identified from this combinatorial library that was not
derived from building blocks 1 or 4 also arrests cell growth in
the G2/M phase (compound 8H, Figure 2B). However, com-
pounds 4A and 1C, derived from the triphenylacetic acid and
triphenylpropionic acid building blocks, respectively, arrest cell
growth in the G1 phase (Figure 2C,D; for percentages at various
cell cycle phases see the Supporting Information).
Effect of TPMAs on Melanoma Cell Lines and NCI
Screening Data. Given the interesting and somewhat unusual
trait of G1 arrest, triphenylmethylamides (TPMAs) 4A and 1C
were tested for their ability to induce death in three melanoma
cell lines: UACC-62 (human), SK-MEL-5 (human), and B16-
F10 (mouse). These melanomas have well-documented and
distinct mutations in their apoptotic pathways, and the two
human cell lines have been particularly well characterized. SK-
MEL-5 expresses very little (less than 15% of melanocyte
controls) Apaf-1,20 a critical protein for apoptosome formation
and caspase-9 activation. The UACC-62 cell line is negative
for expression of p16INK4A and p14ARF;20 these two proteins
are expressed from the INK4A/ARF locus that is deleted in
many melanomas. While the p14ARF gene is totally ablated
from SK-MEL-5, this cell line still expresses the p16INK4A
protein.29 Both SK-MEL-5 and UACC-62 express wild-type p53
(29) Kumar, R.; Sauroja, I.; Punnonen, K.; Jansen, C.; Hemminki, K. Genes
Chromosomes Cancer 1998, 23, 273-277.
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A R T I C L E S
Table 1. Potency of Death Induction for Compounds Identified as Hits in the High-Throughput Screen Depicted in Figure 1a
a IC50 values were determined through analysis of the dose-dependent effect of each compound on U-937 cells in the MTS assay (see graphs in the
Supporting Information). The effect of each compound on the cell cycle was determined by flow cytometric analysis of propidium iodide-stained cells.
protein at normal levels,20 and both have upregulated expression
of ABC multidrug efflux pumps.30 This combination of muta-
tions in critical proteins in the apoptotic cascade and aberrant
expression levels of other proteins enable these melanoma cell
lines to resist the effects of common anticancer agents.
culture.31 Thus bortezomib was used as a control for the toxicity
experiments, and its efficacy is reported in Table 2.
Toxicity of 4A and 1C to Human Bone Marrow. The vast
majority of anticancer drugs are toxic to all rapidly dividing
cell types, leading to deleterious side effects and a reduced
therapeutic window. For instance, hematopoietic bone marrow
is responsible for the production of red and white blood cells,
and the anemia/neutropenia/leukopenia often observed in pa-
tients treated with chemotherapeutic agents is in a large part
due to the toxicity of the anticancer drugs to cells in human
bone marrow.32 Cells obtained from bone marrow of a healthy
human donor were used to determine the relative effect of the
TPMAs on melanoma versus normal cells. Measurement of
compound toxicity to healthy bone marrow cells is a common
method for assessing the selectivity of putative anticancer
agents.33-36 These actively growing cells were treated with
increasing concentrations of compounds 4A and 1C, and cell
viability was evaluated after 72 h, with controls conducted in
the absence of any compound. It was found that while
compounds 4A and 1C are potent inducers of cell death in
melanoma cells, they also have comparable toxicity to these
Evaluation of TPMAs 4A and 1C showed that these
compounds exhibit excellent death-inducing activity in the
melanoma cell lines, with IC50 values ranging from 0.4 to 8.8
µM (Table 2). It was also found that these compounds induce
cell cycle arrest in the G1 phase in these melanoma cell lines
(see Supporting Information). Compounds 4A and 1C were
submitted for testing in the National Cancer Institute’s 60-cell
line screen. The NCI data revealed that the compounds are quite
active against a broad spectrum of cancers and confirmed the
IC50 value of 600 nM against the UACC-62 melanoma cell line
(for full NCI data on compounds 4A and 1C, see the Supporting
Information). The triphenylmethyl group appears crucial to this
activity, as diphenylmethyl derivatives such as compound 3A
show virtually no death induction (Table 2). In addition, the
triphenylacetic and triphenylpropionic acid building blocks
(compounds 1 and 4) have minimal death-inducing activity. As
extensively discussed later, it was determined that the TPMAs
induce apoptosis in these melanoma cell lines. Very recently
the proteasome inhibitor bortezomib (also called VELCADE
or PS-341) was reported to be toxic to melanoma in cell
(31) Amiri, K. I.; Horton, L. W.; LaFleur, B. J.; Sosman, J. A.; Richmond, A.
Cancer Res. 2004, 64, 4912-4918.
(32) Groopman, J. E.; Itri, L. M. J. Natl. Cancer Inst. 1999, 91, 1616-1634.
(33) Konstantinov, S. M.; Topashka-Ancheva, M.; Benner, A.; Berger, M. R.
Int. J. Cancer 1998, 77, 778-786.
(34) de Graaff, M.; Maliepaard, M.; Pluim, D.; Floot, B. J.; Slaper-Cortenbach,
I. C.; Schellens, J. H. Anticancer Drugs 1999, 10, 213-218.
(35) LoRusso, P. M.; et al. InVest. New Drugs 1999, 16, 287-296.
(36) Oredipe, O. A.; Furbert-Harris, P. M.; Laniyan, I.; Griffin, W. M.; Sridhar,
R. Internation. Immunopharm. 2003, 3, 1537-1547.
(30) Szakacs, G.; et al. Cancer Cell 2004, 6, 129-137.
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Figure 2. Analysis of cell populations (after 6 h of treatment) show that compounds such as taxol and 8H arrest growth of U-937 cells in the G2/M phase
of the cell cycle (A and B), whereas amides derived from triphenylacetic acid and triphenylpropionic acid arrest cell growth in the G1 phase of the cell cycle
(C and D).
normal cells isolated from the bone marrow of healthy human
donors (Table 2). It is also worth noting that common anticancer
agents, such as cyclophosphamide37 and etoposide, and the
steroid prednisone (all of which are known S phase arrestors)
have no effect on the melanoma cell lines but are quite toxic to
the bone marrow-derived cells (Table 2). Bortezomib potently
induces cell death in the melanoma cell lines, but is even more
toxic to the bone marrow-derived cells (Table 2).
Second-Generation Library. To minimize general cytotox-
icity and optimize potency for melanoma cells, a second-
generation combinatorial library was constructed. Because
multiple compounds containing amides derived from triphenyl-
acetic and triphenylpropionic acid displayed powerful proapo-
ptotic effects in melanoma cell lines and were shown to induce
apoptosis and arrest the cell cycle in the G1 phase, a second
library was synthesized on a 0.035-mmol scale using only the
triphenylacetic acid (4) and triphenylpropionic acid (1) building
blocks. The acid chlorides derived from the two acids were
treated in parallel with 52 different amines to provide 104 amide
products; this reaction scheme and the various building blocks
are shown in Scheme 2. Again, all of the final products were
purified via filtration through short plugs of silica gel. HPLC
and mass spectrometry were used to characterize the entire
library; in this case 94 of the 104 compounds were found to
have been synthesized in good yields and were isolated in high
purities. The average purity for these 94 compounds was 84%
(see Supporting Information for exact purities of individual
compounds). For IC50 value determinations of the most active
compounds (below), samples utilized were all >97% pure as
1
judged by HPLC analysis and H NMR.
Potency of TPMAs in the Second-Generation Library. All
amides thus produced were evaluated for induction of cell death
in the melanoma cell lines, and the most active compounds then
had their toxicity to the noncancerous bone marrow cells
measured. In general, many of the TPMAs were potent death
inducers in melanoma cell lines, with several displaying
potencies in the 0.5-1.0 µM range. Of the hits from this library,
two compounds in particular, 4AN and 4BI, showed markedly
less (8-12-fold) toxicity to the noncancerous normal cells
(Table 2).
Apoptotic Assays. To determine whether the observed death
induced by the TPMAs was due to apoptosis or necrosis,
common biochemical markers of apoptosis were monitored,
including mitochondrial membrane depolarization, chromatin
(37) Cyclophosphamide is activated in vivo to 4-hydroperoxycyclophosphamide
(4-HC); however, both cyclophosphamide and 4-HC have been used in
cell culture toxicity experiments. For an example of the recent uses of
cyclophosphamide in cell culture experiments, please see: Rzeski, W.; et
al. Ann. Neurol. 2004, 56, 351-360.
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A R T I C L E S
Table 2. IC50 Values for the Induction of Cell Death by Selected TPMAs in Melanoma Cell Lines and Normal Cells from Human Bone
Marrowa
a All IC50 values were determined after a 72-h treatment with the compound. All compounds listed were evaluated at a purity of >97% as assessed by
HPLC (See the Supporting Information).
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Scheme 2. Second-Generation Library of Triphenylmethylamidesa
a The compounds were synthesized in parallel, and after filtration through silica gel the amide products had an average purity of 84%.
condensation, and phosphatidylserine exposure. The depolar-
ization of the mitochondrial membrane facilitates the release
of proapoptotic proteins, such as cytochrome c and AIF
(apoptosis inducing factor) from the mitochondria into the
cytosol.38 Several dyes, including JC-9, have been developed
that provide a sensitive readout of this process.39 As shown in
Figure 3A, treatment of UACC-62 cells with 50 µM of
compound 4BI (the most selective antimelanoma agent of the
TPMAs) shifts the fluorescence of this dye, consistent with
mitochondrial membrane depolarization.
50 µM of 4BI contain highly condensed chromatin (as visualized
with the Hoechst-33258 dye), indicative of apoptosis.
A final event in apoptosis is the exposure of phosphati-
dylserine on the outer leaflet of the cell membrane; this
membrane perturbation allows for recognition of apoptotic cells
by phagocytes, ultimately leading to the engulfment of the
apoptotic cell and the recycling of the various cellular compo-
nents. Phosphatidylserine exposure can be detected by the
binding of fluorescently labeled Annexin V. As shown in Figure
3C, compound 4BI (50 µM) causes apoptosis as measured by
Annexin V staining and flow-cytometry. Compounds 4A and
4AN were also shown to induce apoptotic death in UACC-62,
as evaluated by the three assays described above (see Supporting
Information for images).
Another common biochemical hallmark of apoptosis is
chromatin condensation.38 Certain DNA intercalators exhibit a
greater fluorescence when in the presence of condensed
chromatin and are therefore used as an apoptotic indicator as
measured by flow cytometry and/or microscopy. As seen in
Figure 3B, the nuclei of UACC-62 melanoma cells treated with
TPMA-Treated Cells Have Reduced Levels of Active
NFKB. There is currently no effective therapeutic strategy for
disseminated melanoma, as this cancer is resistant to both
radiation treatments and chemotherapy. As a consequence, the
five-year survival rates for patients with advanced melanoma
(38) Blatt, N. B.; Glick, G. D. Bioorg. Med. Chem. 2001, 9, 1371-1384.
(39) Cossarizza, A.; Baccarani-Contri, M.; Kalashnikova, G.; Franceschi, C.
Biochem. Biophys. Res. Commun. 1993, 197, 40-45.
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A R T I C L E S
of further exploration. Toward this end, the ability of the TPMAs
to affect the cellular level of active NFκB was explored. The
transcription factor NFκB is constitutively active in many
melanoma cell lines, and this activated NFκB translocates to
the nucleus and initiates the transcription of a variety of anti-
apoptotic proteins;41,42 the precise cause of this constitutive
activity is not completely understood. High levels of active
NFκB are known to promote transition from the G1 to the S
phase of the cell cycle by inducing the transcription of cyclin
D1.43 Thus, UACC-62 cells were treated with 4A and the
cellular levels of active NFκB were assessed. As shown in
Figure 4, compound 4A causes a marked decrease in the amount
of active NFκB, as assessed (with an antibody to active NFκB)
by both flow cytometry and microscopy. This reduction of the
levels of active NFκB by 4A may block the progression from
G1 to S phase in melanoma cells and lead to the G1 arrest
observed. As NFκB is constitutively active in many different
cancers,41,42,44 this ability of the TPMAs to cause reduction of
active NFκB is a possible explanation for the general potency
of these compounds in multiple cancer types (as seen through
the NCI screen).
The proteosome inhibitor bortezomib, which also reduces
active levels of NFκB in cancer cells,45 has been clinically
approved for treatment of multiple myeloma.46 Recently,
bortezomib was shown to be effective against melanoma cell
lines and in a mouse model of melanoma.31 Indeed, control
experiments we have performed indicate that bortezomib is quite
toxic to melanoma cell lines (IC50 ) 9-35 nM; Table 2).
However, bortezomib is also extremely toxic to bone marrow
from healthy human donors (IC50 ) 6 pM; Table 2) and is toxic
to mice at fairly low dosages (toxicity at 2 mg/kg with three
times per week dosing45). In contrast, preliminary data indicate
that mice can tolerate daily treatments with 4A at concentrations
as high as 100 mg/kg (unpublished data).
Described herein is the synthesis, identification, and optimi-
zation of several triphenylmethylamides that induce growth
arrest in the G1 phase of the cell cycle and show excellent
potency against three melanoma cell lines in cell culture. Further,
multiple lines of evidence indicate that these compounds induce
apoptosis in melanoma cells. Importantly, these melanoma cell
lines have previously been shown to be resistant to common
anticancer agents and have varied defects in their apoptotic
pathways.20 Additionally, certain TPMAs described herein have
excellent potencies in melanoma cell lines relative to their
toxicity to bone marrow cells derived from healthy human
Figure 3. Induction of apoptosis in UACC-62 (human melanoma) cells
by treatment with 50 µM of compound 4BI (right) versus control (left).
(A) Mitochondrial membrane depolarization (as measured by the dye JC-
9), (B) chromatin condensation (as visualized with Hoechst-33258), and
(C) phosphatidylserine externalization on the surface of UACC-62 cells
(as observed with fluorescently labeled Annexin V) all indicate that 4BI
induces apoptotic cell death.
approaches zero. It is becoming increasingly clear that melanoma
differs significantly at a molecular level from other forms of
cancer. For instance, while a very high percentage (>50%) of
cancers have a mutation in the p53 gene,4 less than 5% of
melanomas have this mutation.40 Instead, recent evidence has
indicated that melanoma cells have defects elsewhere in the
apoptotic machinery.15 For example, certain melanomas have a
methylation-inactivated Apaf-1 signaling complex20 and/or
upregulation of the caspase-inhibiting protein survivin.21 Due
to these distinct differences in the apoptotic proteins, it is not
surprising that drugs effective against other forms of cancer
show no efficacy versus melanoma cells in vitro and in vivo.
Given the potency of the TPMAs against multiple melanoma
cell lines and their unusual G1 arresting properties, their
mechanism of action is potentially novel and therefore worthy
Figure 4. TPMAs reduce the levels of active NFκB in melanoma cells. (A) UACC-62 cells were treated with 50 µM of compound 4A and then stained with
an antibody to active NFκB and analyzed by flow cytometry over a 24-h period. (B) Untreated UACC-62 cells show marked levels of active NFκB when
visualized by microscopy with an antibody to active NFκB. In contrast, UACC-62 cells treated with 50 µM of 4A for 24 h (but still adherent) show very
little active NFκB. Red ) active NFκB, via Cy5-conjugated secondary antibody. Blue ) Hoechst-stained nuclei.
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A R T I C L E S
Dothager et al.
Synthesis of the First-Generation Library of 100 Amides. Amines
A-J were coupled with the 10 acid chlorides derived from acids 1-10
in parallel on a 0.05 mmol scale. Acid chlorides of 1-10 were
synthesized in parallel through addition of 3 equiv of oxalyl chloride
and a catalytic (1 mol %) amount of N,N-dimethylformamide to a stirred
solution of the corresponding carboxylic acid in dry dichloromethane.
After 12 h, solvent and excess oxalyl chloride were removed under
reduced pressure and the resulting acid chlorides were used immediately.
Stock solutions of each amine (0.102 M), each acid chloride (0.17 M),
and triethylamine (0.21 M), to which 5 mol % DMAP was added, were
prepared in dry CH2Cl2. To 100 glass vials was added 0.25 mL of a
stock solution of dry triethylamine (5.3 mg, 0.0525 mmol, 1.05 equiv),
followed by addition of a 0.5-mL aliquot of each amine A-J (0.051
mmol, 1.02 equiv). The vials were capped and cooled in an ice bath to
0-5 °C. At this point, 0.3-mL aliquots of the corresponding acid
chlorides (0.05 mmol) were added, and the vials were capped and
shaken on an orbital shaker at 300 rpm for 48h, during which time the
reactions were allowed to warm to ambient temperature. All reaction
mixtures were diluted with 1 mL of CH2Cl2 and passed through 1.5-
in. silica gel columns packed in Pasteur pipets with hexanes. The
columns were eluted with 2 mL of 1:1/hexanes:ethyl acetate mixture
into tared vials. Solvents were removed under reduced pressure, samples
were dried under vacuum (0.01 mmHg) in a desiccator, and weights
were recorded for all samples. The average isolated yield of the 100
compounds was 94%. Each sample was analyzed by high-throughput
LC/MS (ESI), which allowed for determination of the purity of each
compound and confirmation of each molecular ion peak. The presence
of the molecular ion peak was confirmed for 89 compounds. Com-
pounds 6A-6J failed to be identified by LC/MS and were analyzed
by ESI-MS, 1H NMR, and 13C NMR, all of which confirmed the
formation of the desired product. Compounds 5A-5J were additionally
analyzed by HPLC. Formation of compound 3F could not be confirmed
by any analytical method. The purity of each sample was determined
from the corresponding LC trace (average purity was 89%.)
Synthesis of Second-Generation Library of Triphenylmethyl
Amides. A library of 104 triphenylmethyl amides was generated by
coupling 52 amines with the acid chlorides derived from 1 and 4 in
parallel on a 0.035-mmol scale. The two acid chlorides were synthesized
by known protocols,48,49 recrystallized from hexane, and stored in a
desiccator until use. To each of 104 glass vials was added 0.5-mL
aliquots of a stock solution of dry triethylamine (0.077 M, 0.037 mmol)
and 4-N,N-dimethylaminopyridine (0.004 M, 5 mol %) in dry CH2Cl2.
Aliquots of amine (0.5 mL of a 0.074 M solution, 0.037 mmol) and
acid chloride (0.25 mL of a 0.14 M solution, 0.035 mmol) stock
solutions in dry CH2Cl2 were added to the triethylamine/DMAP
solution, and vials were capped. An additional equivalent of triethyl-
amine was added to those amines that were purchased as the HBr or
HCl salt. Vials were mixed on an orbital shaker at 225 rpm for 48 h.
Crude reaction mixtures were passed through 1.5-in. silica gel columns
packed in Pasteur pipets. Solvent was removed, and weights were
recorded for all isolated products. High-throughput HR-ESI mass
spectrometry was performed on all 104 compounds. Ten compounds
failed to show positive identification (M + 1) as the dominant peak.
These compounds (1AI, 4AI, 1AW, 1AX, 4AX, 1BE, 4BE, 1BR, 1BX,
and 4BX) were not included in yield and purity calculations. The
average isolated yield of the 94 compounds was 77%. Purity (average
purity 84%) was quantified by HPLC. Generally, the 52 amides obtained
from building block 4 were synthesized in higher yields (85% vs 69%)
and purity (90% vs 78%) than those from 1.
donors. These compounds likely function at least in part by
reducing the cellular levels of active NFκB. As the relationship
between G1 cell cycle control and oncogenesis is still ill
defined,47 compounds that arrest cellular growth in this phase
of the cell cycle may have novel macromolecular targets and
can be used to help elucidate the underlying cancer biology. In
addition, such compounds may be effective antimelanoma
agents. Studies to identify the precise molecular target of the
TPMAs and to determine the efficacy of these compounds in
mouse models of melanoma are ongoing and will be reported
in due course.
Experimental Section
Materials. MTS/PMS CellTiter 96 Cell Proliferation Assay reagent
was purchased from Promega (Madison, WI). Cell culture media, Taxol,
poly-L-lysine, and the Cy3 conjugated anti-mouse second antibody were
purchased from Sigma (St. Louis, MO). Alexa Fluor 488 conjugate,
JC-9, propidium iodide, and ProLong Antifade Kit were purchased from
Molecular Probes (Eugene, OR). Microtiter plates and all other materials
were purchased from Fisher (Chicago, IL). The antibody that recognizes
the nuclear localization signal of the p65 subunit of active NFκB was
purchased from Chemicon International (Temecula, CA).
Dry dichloromethane was distilled from P2O5 under dry air atmo-
sphere. Triethylamine was stored over 3-Å molecular sieves. All
reagents were obtained from common commercial sources and used
without further purification.
Compound Analysis. All NMR experiments were recorded in
CDCl3, CD2Cl2, or CD3OD on Varian Unity 400 & 500 MHz
spectrometers with residual undeuterated solvent as an internal refer-
ence. Chemical shift, δ (ppm); coupling constants, J (Hz); multiplicity
(s ) singlet, d ) doublet, t ) triplet, q ) quintet, m ) multiplet); and
integration are reported. High-resolution mass spectral data were
recorded on a Micromass Q-Tof Ultima hybrid quadrupole/time-of-
flight ESI mass spectrometer at the University of Illinois Mass
Spectrometry Laboratory. Infrared spectra were recorded on a Perkin-
Elmer Spectrum BX spectrophotometer, referenced to a polystyrene
standard, and the peaks are reported in cm-1. Solvents for chromatog-
raphy were reagent grade and were used without further purification.
Silica gel chromatography was performed on EMD Biosciences silica
gel 60 (230-400 mesh). Thin-layer chromatography plates (Merck,
245 nm fluorescent indicator) were visualized by UV and stained with
cerium ammonium molybdate (CAM), iodine (I2), ninhydrin, or DPIP
(0.5 mg 2,6-dichloroindophenolate hydrate per mL in EtOH). Purified
products were analyzed on a Varian 2510 HPLC equipped with an
Alltech Alltima C18 column (20 mm × 2.1 mm × 3 µm) with an
acetonitrile/0.1% trifluoroacetic acid (in Millipore MilliQ-filtered water)
solvent gradient. Eluted solute was detected with a Waters 486 UV
multiple wavelength absorbance detector operated at 254 nm. All acid
and amine building blocks were used as received from common
commercial suppliers, except 8 and 9, which were prepared by
Wadsworth-Emmons olefination following a procedure described in
Organic Synthesis (Collect. Vol. 5, p 547) from commercially obtained
m-anisaldehyde and 4-methoxynaphthalene-1-carbaldehyde, respec-
tively.
(40) Satyamoorthy, K.; Bogenrieder, T.; Herlyn, M. Trends Mol. Med. 2001, 7,
191-194.
(41) Monks, N. R.; Biswas, D. K.; Pardee, A. B. J. Cell Biochem. 2004, 92,
646-650.
(42) Aggarwal, B. B. Cancer Cell 2004, 6, 203-208.
(43) Joyce, D.; Albanese, C.; Steer, J.; Fu, M.; Bouzahzah, B.; Pestell, R. G.
Cytokine Growth Factor ReV. 2001, 12, 73-90.
Cell Culture Conditions. U-937, HL-60, SK-MEL-5, and UACC-
62 cells were grown in RPMI 1640 media supplemented with 10%
FBS, B16-F10 cells were grown in Eagle’s minimal essential medium
with Earle’s BSS supplemented with 10% FBS, and human bone
(44) Bours, V.; Dejardin, E.; Goujon-Letawe, F.; Merville, M. P.; Castronovo,
V. Biochem. Pharmacol. 1994, 47, 145-149.
(45) Sunwoo, J. B.; Chen, Z.; Dong, G.; Yeh, N.; Crowl Bancroft, C.; Sausville,
E.; Adams, J.; Elliott, P.; Van Waes, C. Clin. Cancer Res. 2001, 7, 1419-
1428.
(46) Rajkumar, S. V.; Richardson, P. G.; Hideshima, T.; Anderson, K. C. J.
Clin. Oncol. 2005, 23, 630-639.
(47) Massague, J. Nature 2004, 432, 298-306.
(48) Seto, C. T.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 905-916.
(49) Bollyky, L. J.; Whitman, R. H.; Clarke, R. A.; Rauhut, M. M. J. Org.
Chem. 1967, 32, 1663-1667.
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8694 J. AM. CHEM. SOC. VOL. 127, NO. 24, 2005

Arrest of Melanoma Cell Growth in the G1 Phase
A R T I C L E S
marrow cells were grown in Iscove’s modified Dulbecco’s medium
supplemented with 40% FBS. All cell lines were incubated at 37 °C in
a 5% CO2, 95% air atmosphere. U-937 and HL-60 cells were split every
2-3 days as needed and UACC-62 and B16-F10 cells were split when
they reached approximately 80% confluency at a 1:6 ratio.
cells and the absorbance was measured at 520 nm in a Spectra Max
Plus 384 plate reader (Molecular Devices, Sunnyvale CA).
Annexin V staining. UACC-62 cells (10 mL) were seeded into a
12.5 cm2 flask and grown to confluency. The media was replaced with
media containing the test compounds or vector only. The cells were
incubated for various times, trypsinized, and harvested by centrifugation.
The cells were then washed in PBS buffer before staining. After PBS
washes, the cells were washed in 1 mL of Annexin V binding buffer
(10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) and
resuspended in 100 µL of Annexin V binding buffer. Then 5 µL of
Annexin V, Alexa Fluor 488 conjugate was added, and the tubes were
incubated at room temperature for 15 min protected from light. Annexin
V binding buffer (400 µL) was then added, followed by the addition
of 1 µL of a 1 mg/mL solution of propidium iodide. The fluorescent
intensity of each cell was determined by flow cytometry at 525 nm
(green channel) and 675 nm (red channel). At least 10 000 cells were
analyzed in each experiment.
Cell Death Assay and IC50 Value Determination in White Blood
Cells. To each well of a 96-well flat-bottom microtiter plate was added
50 µL RPMI-1640 + 10% FBS media. During the initial screen, library
compounds dissolved in DMSO were added to the plates to give a 50
µM solution. To determine the IC50 values, active compounds were
added to a 96-well plate in varying concentrations. Controls were
performed in which only DMSO (containing no compound) was
transferred into wells. U-937 or HL-60 cells were harvested by
centrifugation at 250g for 5 min. Cells were then resuspended in RPMI-
1640 + 10% FBS, counted using a hemocytometer and diluted to 8 ×
105 cells/mL. A 50-µL aliquot of the cell solution was then added to
each well. The cells were then incubated with the compounds for 24
or 72 h. Cell death was quantitated by adding 20 µL of the MTS/PMS
CellTiter 96 Cell Proliferation Assay reagent to each well. The plates
were incubated at 37 °C for approximately 1 h until the colored product
formed. The absorbance was then measured at 490 nm in a Spectra
Max Plus 384 plate reader (Molecular Devices, Sunnyvale CA).
Mitchondrial Membrane Potential. UACC-62 cells (10 mL) were
seeded into a 12.5 cm2 flask and grown to confluency. The media was
replaced with media containing the test compounds or vector only. The
cells were incubated for various times, trypsinized, and harvested by
centrifugation. The cells were then washed in PBS buffer before
staining. After PBS washes, the cells were resuspended in 1 mL of
PBS. JC-9 dye (10 µg) was added, and the cells were incubated at
room temperature for 10 min protected from light. The cells were then
washed two times with PBS and brought up in 500 µL of PBS. The
fluorescent intensity of each cell was determined by flow cytometry at
525 nm (green channel) and 675 nm (red channel). At least 10 000
cells were analyzed in each experiment.
Cell Cycle Analysis. For suspension cells, 500 µL of RPMI-1640
+ 10% FBS media was added to each well of a 24-well plate. Test
compounds and Taxol were added to the plate in varying concentrations.
Controls were performed in which only the vector (DMSO or ethanol)
was transferred into wells. U-937 cells were harvested by centrifugation
at 250g for 5 min. Cells were then resuspended in RPMI-1640 + 10%
FBS, counted using a hemocytometer, and diluted to 2 × 106 cells/
mL. Then 500 µL of the cell solution was added to each well, bringing
the final concentration of cells to 1 × 106 cells per well. The cells
were then incubated with the compounds for 6 h and harvested by
centrifugation.
NFKB Staining. UACC-62 (10 mL) cells were seeded into a 12.5
cm2 flask and grown to confluency. The media was replaced with media
containing the test compounds or vector only. The cells were incubated
for various times, scraped with a rubber policeman, and harvested by
centrifugation. The cells were then washed twice in ice-cold PBS buffer
before fixing at -20 °C in 100% ethanol. The fixed cells were incubated
overnight at 4 °C, followed again by washing twice in PBS buffer.
The cells were then resuspended in 100 µL of PBS buffer containing
10 µg/mL of an antibody that recognizes active NFκB. After incubation
at room temperature for 2 h followed by two washes in PBS buffer,
the cells were then resuspended in 1.0 mL of a 1:5000 dilution of an
anti-mouse second antibody conjugated to Cy5 and were incubated at
room temperature in the dark for 2 h. The cells were then washed twice
in PBS buffer and resuspended in 400 µL of PBS buffer. The fluorescent
intensity of each cell was determined by flow cytometry at 675 nm
(red channel). At least 50 000 cells were analyzed in each experiment.
Microscopy of Condensed Chromatin and NFKB. To observe
chromatin condensation, 1 mL of UACC-62 cells was seeded into a
24-well plate and grown to confluency. The media was replaced with
media containing the test compounds or vector only. The cells were
incubated for 24 h followed by resuspension by pipeting vigorously
up and down. The cells were removed from the media by centrifugation.
The cells were then washed in PBS buffer before being plated onto
polylysine-coated no. 1 thickness coverslips. The coverslips were then
incubated at 37 °C in a humidified incubator for 1 h. The coverslips
with adhered cells were washed twice with PBS, fixed at 37 °C in 4%
formaldehyde in BRB80 buffer (80 mM Pipes, pH 6.9; 1 mM EGTA;
1 mM MgCl2) for 30 min and then rinsed again twice with PBS. Cells
were permeabilized in PBS containing 0.5% Triton X-100 for 10 min
followed by rinsing three times with PBS containing 0.1% Triton X-100.
Coverslips were incubated with 2 µg/mL Hoechst-33258 in PBS for
30 min at room temperature. Coverslips were rinsed twice with PBS
and mounted on glass slides using a ProLong Antifade Kit. Images of
chromatin were obtained at 400× magnification on a Zeiss Axiovert
100 microscope. For immunofluorescence of NFκB, UACC-62 cells
were grown on glass coverslips by adding 2 mL of cells to a 35 × 10
mm style tissue culture dish containing a single coverslip. The media
For adherent cells, 1 mL of cells was seeded into the wells of a
24-well plate and grown to confluency. The media was replaced with
media containing the test compounds or vector only. The cells were
incubated with the compounds for various times, trypsinized, and
harvested by centrifugation. The cells were then washed with PBS and
resuspended in ice-cold 100% ethanol. After being harvested the cells
were then stored at 4 °C overnight. The cells were pelleted out of the
ethanol by centrifugation, washed in PBS, and resuspended in 50 µL
PBS containing 100 µg/mL RNase A. The cells were incubated at 4
°C for 4 h. A 400-µL aliquot of a 50 µg/mL solution of propidium
iodide was then added, and the cells were analyzed by flow cytometry.
Cell Death Assay and IC50 Value Determination in Melanoma
Cells. To each well of a 96-well flat-bottom microtiter plate was added
50 µL of RPMI-1640 + 10% FBS or 50 µL of MEM + 10% FBS
media. During the initial screen, library compounds dissolved in DMSO
were added to the plates to give a 1 µM solution. To determine the
IC50 values, active compounds were added to a 96-well plate in varying
concentrations. Controls were performed in which only DMSO
(containing no compound) was transferred into wells. UACC-62, SK-
MEL-5, or B16-F10 cells were trypsinized and harvested by centrifuga-
tion at 250g for 5 min. Cells were then resuspended in the appropriate
media, counted using a hemocytometer and diluted to 1 × 105 cells/
mL. Then 50 µL of the cell solution was added to each well. The cells
were then incubated with the compounds for 72 h. Cell death was
quantitated by using sulforhodamine B. To accomplish this, the media
was replaced with 200 µL of 10% trichloroacetic acid. The plate was
incubated for 1 h at 4 °C. The cells were then washed five times with
distilled water, and 200 µL of a 0.4% sulforhodamine B in a 1% acetic
acid solution was added to the plate. The plate was then incubated for
20 min at room temperature. The plate was then washed five times
with 1% acetic acid, and 200 µL of a 10 mM solution of unbuffered
Tris was added. The plates were shaken to release the dye from the
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J. AM. CHEM. SOC. VOL. 127, NO. 24, 2005 8695

A R T I C L E S
Dothager et al.
was replaced with media containing the test compounds or vector only
and incubated for 24 h. The coverslips with adhered cells were removed
from the media and washed twice with PBS, fixed at 37 °C in 4%
formaldehyde in BRB80 buffer (80 mM Pipes, pH 6.9; 1 mM EGTA;
1 mM MgCl2) for 30 min, and then rinsed again twice with PBS. Cells
were permeabilized in PBS containing 0.5% Triton X-100 for 10 min
followed by rinsing three times with PBS containing 0.1% Triton X-100.
The cells on coverslips were then incubated for 10 min in PBS
containing 2% BSA followed by incubation for 2 h in 10 µg/mL of
anti-active NFκB antibody. The cells were then washed three times in
PBS containing 0.1% Triton X-100 followed by a 2 h incubation in a
1:5000 dilution of anti-mouse second-antibody conjugated with Cy5.
The cells were then washed three times with PBS containing 0.1%
Triton X-100 and then incubated with 2 µg/mL Hoechst-33258 in PBS
for 30 min at room temperature. Coverslips were rinsed twice with
PBS and mounted on glass slides using a ProLong Antifade Kit.
Fluorescent images of NFκB and Hoechst stained cells were obtained
at 400× magnification on a Zeiss Axiovert 100 microscope.
Acknowledgment. We thank the University of Illinois for
support of this research and Prof. Ronald Hoffman (University
of Illinois-Chicago Cancer Center) for the generous gift of the
human bone marrow. We acknowledge the assistance of the
Flow Cytometry Facility of the Biotechnology Center, Univer-
sity of Illinois. We thank Millennium Pharmaceuticals for the
gift of bortezomib. We thank Dr. Vera Mainz and Furong Sun
of the University of Illinois School of Chemical Sciences Mass
Spectrometry Laboratory for assistance with mass spectral
analysis. The Q-Tof Ultima mass spectrometer was purchased
in part with a grant from the National Science Foundation,
Division of Biological Infrastructure (DBI-0100085). P.J.H. is
a fellow of the Alfred P. Sloan foundation.
Supporting Information Available: Tables listing purity of
library members, characterization data for individual compounds,
dose-response graphs used in IC50 determinations, NCI data,
apoptotic data not shown in the text, and complete author lists
for references with 10 or more authors. This material is available
free of charge via the Internet at http://pubs.acs.org.
Data Analysis. The data from all flow cytometry experiments was
analyzed using Summit Software (Cytomation, Fort Collins, CO) and
ModFit software (Verity Software House, Inc., Topsham, ME). IC50
values were determined using Table Curve (Systat, Richmond, CA)
using a logistic dose-response model.
JA042913P
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8696 J. AM. CHEM. SOC. VOL. 127, NO. 24, 2005