Triphenylmethylamides (TPMAs): Structure-activity relationship of compounds that induce apoptosis in melanoma cells

Article abstract of DOI:10.1016/j.bmcl.2008.07.128

Triphenylmethylamides (TPMAs) have been previously identified as compounds that arrest cells in the G1-phase of the cell cycle and induce apoptotic death in melanoma cell lines in culture. Here we report the synthesis of a series of TPMA derivatives, allo

Full text of DOI:10.1016/j.bmcl.2008.07.128

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5888–5891
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Triphenylmethylamides (TPMAs): Structure–activity relationship
of compounds that induce apoptosis in melanoma cells
*
Rahul Palchaudhuri, Paul J. Hergenrother
Department of Chemistry, Roger Adams Laboratory, University of Illinois at Urbana–Champaign, Urbana, IL 61801, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Triphenylmethylamides (TPMAs) have been previously identified as compounds that arrest cells in the
G1-phase of the cell cycle and induce apoptotic death in melanoma cell lines in culture. Here we report
the synthesis of a series of TPMA derivatives, allowing the structure–activity relationship of this class of
molecules to be established. Several new compounds have been identified that induce death in UACC-62
and SK-MEL-5 human melanoma cell lines, including a compound with enhanced aqueous solubility.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 6 June 2008
Revised 25 July 2008
Accepted 29 July 2008
Available online 3 August 2008
Keywords:
Pro-apoptotic
Cancer
Anticancer drug
Anticancer compound
Apoptosis
Melanoma
Metastatic melanoma is a particularly aggressive form of cancer
that is resistant to standard anticancer therapies. If identified early,
melanoma (Stage I/II) primary tumors can be surgically resected
with a >95% success rate.1 In contrast, late-stage (Stage IV) meta-
static melanoma is one of the most deadly forms of cancer, with
the median survival of patients with distant metastases being 7–
8 months.2 The current standard of care for patients with late-stage
melanoma is dacarbazine/temozolomide chemotherapy or immu-
the treatment of certain melanomas (and other cancers).11,12 To-
wards this end, we recently identified the triphenylmethylamides
(TPMAs) as a novel class of molecules that induce G1-phase arrest
and apoptosis in several human melanoma cell lines in culture.11
Two representative TPMAs, 4A and 4BI, are shown in Figure 1.
These compounds also reduce cellular levels of the transcription
factor NFj
B in melanoma cell lines in culture.11 In addition, the
compound 4BI is relatively non-toxic against bone marrow cells
from healthy human donors,11 and [11C]labeled TPMAs have been
synthesized for positron emission tomography (PET) imaging.13
While the functionality on the nitrogen side of the amide has little
influence on the potency of the TPMAs (amides based on pheneth-
ylamine, 4-methoxy-phenylamine, and 2-(3-methoxy-phenyl)-
ethylamine are all potent inducers of apoptosis), the triphenyl-
methyl moiety is critical to activity as the diphenylmethylamides
are largely inactive.11
notherapy involving interleukin-2/interferon-a 3–5
Few patients
.
respond positively to these treatments; sometimes tumor shrink-
age is observed for a few months, after which tumor growth typi-
cally resumes.5,6 Clearly, novel compounds and new biological
targets are needed to address the limitations of current melanoma
therapies.
Drugs that target rapidly dividing cells by interfering with DNA
synthesis (S-phase arrest) or mitosis (M-phase arrest) such as tax-
ol, etoposide, cisplatin, and doxorubicin have failed to demonstrate
efficacy in large randomized trials with melanoma patients.7 The
resistance to DNA-targeted therapy may be due to down-regula-
tion of DNA mismatch repair enzymes in melanoma.8 Additionally,
elevated levels of survivin, a protein that assists mitotic spindle
formation permitting the evasion of the G2/M checkpoint, may al-
low melanoma to resist the effect of anti-mitotics.9,10 Given these
characteristics, we hypothesized that compounds inducing arrest
in the G1-phase of the cell cycle might prove to be effective for
Herein, we report our effort to optimize the anticancer proper-
ties of the TPMAs and to further define their structure–activity
relationship (SAR). This study was performed through synthesis
* Corresponding author.
Figure 1. The TPMAs 4A and 4BI were previously identified as potent inducers of
E-mail address: hergenro@uiuc.edu (P.J. Hergenrother).
cell death in human melanoma cell lines in cell culture.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.128

R. Palchaudhuri, P. J. Hergenrother / Bioorg. Med. Chem. Lett. 18 (2008) 5888–5891
5889
of derivatives with functionalized triphenylmethyl moieties and
extensions of the carbon chain between the triphenylmethyl moi-
ety and the amide. Various triphenylmethyl-containing carboxylic
acids were prepared (as described in Supporting information) and
the length of the carbon chain between the triphenylmethyl and
the amide reduced the potency against UACC-62 cells while the
activity against SK-MEL-5 cells remained unchanged as observed
for 10-1-BI, 10-2-A, 10-2-BI, 10-3-A, and 10-3-BI.
coupled with phenethylamine or
D
-phenylalanine methyl ester
It was of interest to determine whether the TPMA derivatives
share a similar mode of action to the unfunctionalized TPMAs 4A
and 4BI, as it was recently shown that anticancer compounds with
the triphenylmethyl motif can be placed into at least four different
categories based on their mechanisms of action, with position of
cell cycle arrest being a major discriminator.12 Thus, several of
the most potent compounds identified herein were tested for their
ability to induce cell cycle arrest. For this assay, the rapidly divid-
ing HL-60 cell line (human leukemia) was utilized. These cells were
using benzotriazol-1-yloxytripyrrolidinophosphonium hexafluoro-
phosphate (PyBOP)-mediated coupling reactions (Chart 1).
The desired TPMAs were obtained in moderate to excellent
yields (72% average yield). The compounds were named based on
the triphenylmethyl (first number), the number of methylenes be-
tween the triphenylmethyl and the amide (second number), and
whether they were derivatives of phenethylamine or D-phenylala-
nine (A or BI). The TPMA derivatives synthesized were evaluated in
cell culture against two human melanoma cell lines, SK-MEL-5 and
UACC-62. Cells were incubated with the compound (at a range of
concentrations) for 72 h, at which time growth inhibition was as-
sessed by quantification of biomass using the sulforhodamine B as-
say.14 To obtain the IC50 values, the compounds were assessed on
three different occasions and the average IC50 values and standard
deviations were determined (see Supporting information for dose–
response curves).
As shown in Chart 1, introduction of functional diversity in the
triphenylmethyl group considerably affected the biological proper-
ties of the TPMAs. With the exception of 5-0-A, hydroxyl-bearing
TPMAs (such as 4-0-A, 4-0-BI, 6-0-A, 6-0-BI, 7-0-A and 7-0-BI)
were less potent than the unfunctionalized TPMAs 4A and 4BI de-
spite being more soluble in aqueous solution.
Methyl ether-substituted TPMAs, 1-0-A and 1-0-BI, were
slightly less potent than the unfunctionalized TPMAs 4A and 4BI.
Increasing the number of methyl ether substitutions reduced the
potency as evidenced by 2-0-A, 2-0-BI, 3-0-A, and 3-0-BI. TPMAs
9-0-A and 9-0-BI bearing conformationally rigid triphenylmethyl
motifs were up to 4-fold less active than 4A and 4BI, highlighting
the importance of flexibility in the triphenylmethyl motif. Replace-
ment of one of the phenyls with a 2-thiophene (8-0-BI and 8-0-A)
resulted in a moderate decrease in potency. Curiously, increasing
treated with 20 lM compound for 6 h, after which the percentage
cell population distributed in the various phases of the cell cycle
was determined using cell flow cytometry and propidium iodide
staining. As shown in Figure 2, the TPMAs 1-0-A, 2-0-A, 5-0-A,
70
G0/G1
S
60
50
40
30
20
10
0
G2/M
Control 4A
4BI
1-0-A
2-0-A
5-0-A 10-3-A
Figure 2. Cell cycle analysis of HL-60 cells treated with various TPMAs. Cells were
incubated for 6 h in the presence of 20 M compound, and then analyzed by
l
propidium iodide staining and cell flow cytometry. Control indicates cells treated
with vehicle only.
Chart 1. Synthesis and biological evaluation of TPMA phenethylamine (A) and
D-phenylalanine (BI) derivatives with functionalized triphenylmethyl groups and varying
carbon chain lengths between the triphenylmethyl motif and amide. The values for phenethylamine derivatives are italicized while those for D-phenylalanine derivatives are
in parentheses. Compounds 4A and 4BI were synthesized as previously reported.11 Average IC50 values and standard deviations were determined from three independent
experiments. Biomass was assessed by the sulforhodamine B assay after 72 h incubation in the presence of compound.

5890
R. Palchaudhuri, P. J. Hergenrother / Bioorg. Med. Chem. Lett. 18 (2008) 5888–5891
Figure 3. The TPMA 5-0-A induces cell death via apoptosis. U-937 cells were treated with vehicle (left) or 100
lM 5-0-A (right) for 48 h, and the cell populations were
quantified using FITC-conjugated Annexin V (FITC-A) and propidium iodide (PI-A) staining together with cell flow cytometry.
and 10-3-A all induce G1-phase arrest, similar to the parent TPMAs
4A and 4BI.
inducing cell death in SK-MEL-5 cells as 4A and 4BI. This work adds
to a growing body of literature showing that a variety of com-
pounds possessing the triphenylmethyl group induce death in can-
cer cells, albeit by diverse mechanisms.11,12,15–22 While the
biological target of the TPMAs remains unknown, the structure–
activity relationship described herein may prove useful for further
optimization and development of triphenylmethyl-containing
anticancer agents.
The TPMA 5-0-A, which is slightly more potent than the unfunc-
tionalized TPMAs 4A and 4BI, was further evaluated for its ability
to induce apoptotic cell death as assessed by the externalization
of phosphatidylserine that occurs prior to disruption of cell mem-
brane integrity. Phosphatidylserine externalization was assessed
by binding of FITC-conjugated Annexin V, and cell membrane
integrity was assessed by exclusion of propidium iodide as mea-
sured by flow cytometry. As shown in Figure 3, U-937 human lym-
Acknowledgments
phoma cells treated for 48 h with 100 lM of TPMA 5-0-A show a
large shift to the early apoptotic quadrant (lower right; Annexin
V positive/propidium iodide negative) indicating that 5-0-A-in-
duced G1-phase cell cycle arrest leads to apoptosis.
In order to assess whether the TPMAs 4A, 4BI, 5-0-A, and 10-2-
BI possess general anticancer properties, their ability to affect the
viability of various cancer cell lines in cell culture was evaluated.
As shown in Table 1, the TPMAs exhibited potent anti-proliferative
activity against leukemia, lymphoma, and breast cancer cell lines
in addition to melanoma. In contrast, the rat adrenal cancer PC-
12 cell line was found to be fairly resistant to the effects of the
We acknowledge the assistance of the cell flow cytometry and
mass spectrometry facilities at the University of Illinois, Urbana–
Champaign. We are grateful to the American Cancer Society
(RSG-07-026-01) for funding this work.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.07.128.
TPMAs with 5-0-A being the most potent (IC50 = 16
4 lM).
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5-0-A
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UACC-62
HL-60
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PC-12
5.7 1.7
14
6.2 0.5
5.9 0.3
8.1 1.1
85 14
4.2 2.6
2.1 1.2
7.3 1.2
7.2 1.8
8.0 1.4
2.5 0.9
40
Melanoma (human) 8.3 2.8
Leukemia (human) 7.7 1.6
Lymphoma (human) 9.5 1.1
Breast (human)
Adrenal (rat)
2
5
6.0 1.5
6.8 3.2
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7.1 1.0
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