Synthesis and cytotoxicity of 1-phenylethanolamine carboxamide derivatives: Effects on the cell cycle

Article abstract of DOI:10.1007/s00044-009-9258-9

Seven novel analogues of 1-phenylethanolamine carboxamide derivatives, 3a-3g, related to carboxamides isolated from Isodon excisus were synthesized and evaluated for their cytotoxic and apoptosis-induction properties against murine B16 and leukemia L1210 cell lines. Compounds containing no substitution at the 4′-position (3a-3d) or containing a 4′-amino (3e-3g) group were investigated. Generally, the amino-containing compounds were slightly more active than their unsubstituted congeners. Also, the indole-containing compounds 3c and 3f gave the strongest cytotoxic activity (IC₅₀ = 25-87 μM) against the growth of L1210 and B16 cancer cells. Compound 3f was subjected to flow cytometry studies and it was found to induce L1210 cells grown in culture to undergo apoptosis.

Full text of DOI:10.1007/s00044-009-9258-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2010) 19:1141–1152
DOI 10.1007/s00044-009-9258-9
ORIGINAL RESEARCH
Synthesis and cytotoxicity of 1-phenylethanolamine
carboxamide derivatives: effects on the cell cycle
•
Balaji Babu Lori Forrest Paul Weisbruch
•
•
•
Sameer Chavda Hari Pati Moses Lee
•
Received: 20 May 2009 / Accepted: 10 September 2009 / Published online: 6 October 2009
¨
Ó Birkhauser Boston 2009
Abstract Seven novel analogues of 1-phenylethanolamine carboxamide deriva-
tives, 3a–3g, related to carboxamides isolated from Isodon excisus were synthesized
and evaluated for their cytotoxic and apoptosis-induction properties against murine
B16 and leukemia L1210 cell lines. Compounds containing no substitution at the
40-position (3a–3d) or containing a 40-amino (3e–3g) group were investigated.
Generally, the amino-containing compounds were slightly more active than their
unsubstituted congeners. Also, the indole-containing compounds 3c and 3f gave the
strongest cytotoxic activity (IC50 = 25–87 lM) against the growth of L1210 and
B16 cancer cells. Compound 3f was subjected to flow cytometry studies and it was
found to induce L1210 cells grown in culture to undergo apoptosis.
Keywords Phenylethanolamine ꢀ Carboxamide ꢀ Cancer ꢀ Cytotoxicity ꢀ
Apoptosis ꢀ Synthesis
Introduction
Apoptosis, or programmed cell death, is a highly conserved process used by
multicellular organisms to rid themselves of unwanted or damaged cells (Hipfner
and Cohen, 2004). Hallmarks of apoptosis include cellular membrane blebbing,
B. Babu ꢀ S. Chavda ꢀ M. Lee (&)
Division of Natural and Applied Sciences, Department of Chemistry, Hope College, Holland,
MI 49423, USA
e-mail: lee@hope.edu
L. Forrest ꢀ P. Weisbruch ꢀ H. Pati ꢀ M. Lee
Department of Chemistry, Furman University, Greenville, SC 29613, USA
H. Pati
Department of Chemistry, Sambalpur University, Jyoti Vihar, Orissa 768 019, India

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Med Chem Res (2010) 19:1141–1152
cleavage of certain nucleases and polymerases, and activation of cysteine proteases
known as caspases. From a medicinal chemistry perspective, small molecules that
either inhibit or induce apoptosis have potential in the development of novel
therapeutic agents, especially for the treatment of cancer (Sluyser, 2005; Narayan
et al., 2003; Du et al., 2006; Hansch et al., 2003; Ghobrial et al., 2005).
The focus of this article is the development of small molecules that have the
property to induce cancer cells to undergo apoptosis, as it potentially relates to the
control of cancerous growth and treatment. Examples of methyl ethers of
phenylethanolamine carboxamide derivatives, such as compound 1a (Fig. 1),
isolated from Isodon excisus, which protected U937 lymphoma cells from
etoposide-induced apoptosis, have recently been reported (IC50 = 10.2 lg/ml)
(Lee et al., 1999, 2001). Isodon excisus grows widely in Korea, and aerial parts of
the plant are used for detoxification (Lee et al., 1999, 2001). Due to their intriguing
influence on the cell cycle, this class of compounds was studied further. The results
showed that a few of the compounds significantly inhibited apoptosis in cancer cells
(Xing et al., 2003). Additionally, related b-aryl ethanolamine derivatives were also
found to have potential application in combating hypertension, arrthymia, asthma,
and angina (Buchanan et al., 2004). Interestingly, nonmethyl ether analogues of
phenylethanolamine carboxamide derivatives were recently discovered by Nest-
erenko’s group (2003) to exhibit proapoptotic properties. Specifically the nonmethyl
40-hydroxyphenylethanolamine cinnamoyl carboxamide 1b (Fig. 1) was found to
exhibit strong proapoptotic cytotoxic activity against cancerous U937 as low as
IC50 = 44 lg/ml, yet it was noncytotoxic against normal splenocytes (Nesterenko
et al., 2003). Based on this background, seven nonmethyl ethers of 1-phenyleth-
anolamine arylcarboxamides that were either unsubstituted at the 40-position or
contained a 40-amino group were designed and synthesized. The 40-amino analogues
have not been studied previously and these new compounds are attractive targets
because the amino moiety is potentially beneficial toward enhancing water
solubility as well as providing a nucleophilic handle for further derivatization.
Experimental
Method A: for preparation of 2a and 2b
To a solution of benzaldehyde (1 eq) and nitromethane (1.2 eq) in dry acetonitrile
was added powdered potassium phosphate (0.1 eq). The solution was stirred at room
(a)
(b)
OH
OH
OH
OMe
H
H
N
N
OCH3
OCH3
O
O
HO
HO
Fig. 1 Structure of the naturally occurring methyl ether of 1-phenylethanolamine carboxamide, 1a, and
its hydroxylated analogue, 1b

Med Chem Res (2010) 19:1141–1152
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temperature overnight. The solvent was removed in vacuo and the residue was
extracted in ethyl acetate and washed with water. The aqueous layer was neutralized
to pH 4 and the organic layer was separated, dried over Na2SO4, filtered, and
removed under vacuum to give residue, which was purified by column chroma-
tography using 0–30% ethyl acetate/hexanes to get the desired product.
Compound 2a
1
Yield = 66%; Rf = 0.5 (20% EtOAc/hexanes). H-NMR: d (500 MHz, CDCl3) d
7.88 (dd, J 1.5, 7.5, 2H), 7.64 (dt, J 1.5, 7.0, 1H), 7.54 (t, J 8.0, 2H), 5.46 (dd, J 3.0,
9.5, 1H), 4.64–4.50 (m, 2H).
Compound 2b
1
Yield = 86%; Rf = 0.2 (20% EtOAc/hexanes). H-NMR: d (500 MHz, CDCl3) d
8.26 (dd, J 2.0, 7.0, 2H), 7.63 (d, J 9.0, 2H), 5.61 (dd, J 4.0, 9.0, 1H), 4.59 (d, J
4.0, 2H).
Method B: for preparation of 3a, 3c, 3d, 3e, and 3g
The nitro compound (1.0 g) was reduced using 100%, by wt, 5% Pd/C and 75 ml
chilled methanol under H2. After 18 h, the catalyst was removed by filtration over
celite and rinsed with methanol. The methanol was removed under reduced pressure
and the residue was coevaporated with dry methylene chloride (3 9 5 ml). The
resulting amine was dissolved in dry pyridine (10 ml) and dry triethylamine
(0.3 ml).
To the corresponding carboxylic acid (1.29 mmol) dissolved in dry methylene
chloride (10 ml) was added 1 ml thionyl chloride and the mixture was refluxed for
1.5 h until the solid dissolved completely. The thionyl chloride was removed via
aspiration under heat and coevaporated twice with methylene chloride. The acid
chloride (1.29 mmol) was redissolved in dry methylene chloride (5 ml) and added
dropwise to the amine solution kept at 0°C. The reaction was stirred at the same
temperature for 15 min, then brought to room temperature and stirred overnight.
The solvent was removed under reduced pressure and the product was redissolved in
CHCl3 and washed with water. The organic solution was dried over Na2SO4 and the
solvent removed under vacuum. The residue was purified by silica gel column
chromatography using methanol and chloroform.
Compound 3a
Light-orange solid; yield = 63%; MP = 112–115°C, Rf = 0.3 (20% EtOAc/
hexanes). IR cm-1 (neat): 3367, 3288, 2932, 1693, 1649, 1586, 1490, 1452,
1316, 1292, 1255, 1163, 1096, 1037, 982, 842, 768, 702, 673. 1H-NMR: d
(500 MHz, CDCl3) 7.40 (d, J 13.5, 1H), 7.30 (d, J 5.5, 1H), 7.30–7.18 (m, 3H), 7.02
(d, J 6.5, 2H), 6.98 (t, J 11.5, 1H), 6.79 (d, J 3.0, 1H), 6.48 (t, J 11.0, 2H), 4.87 (m,

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Med Chem Res (2010) 19:1141–1152
1H), 3.74 (s, 3H), 3.39–3.37 (m, 1H), 3.32–3.29 (m 1H). MS (direct probe: ES?):
calc. mass = 297, actual mass = 298 (M ? H).
Compound 3c
Light-brown solid; yield = 27%; MP = 120–130°C; Rf = 0.3 (20% EtOAC/
hexanes). IR cm-1 (neat): 3305, 2921, 1698, 1651, 1627, 1531, 1452, 1215,
1028, 805, 756, 701. 1H-NMR d (500 MHz, CDCl3): 7.88 (d, J 8.5, 2H), 7.68 (d, J
8.0, 1H), 7.46 (d, J 8.0, 1H), 7.22 (t, J 8.5, 1H), 7.08 (d, J 2.0, 1H), 6.97 (t, J 5.0,
2H), 6.83 (d, J 2.0, 1H), 4.45 (m, 1H), 4.00 (m, 1H), 3.71 (s, 3H), 3.58 (m, 1H). MS
(direct probe: ES?): calc. mass = 310, actual mass = 311 (M ? H).
Compound 3d
Light-orange solid; yield = 75%; MP = 198–200°C, 148°C (dec.); Rf = 0.8 (20%
EtOAc/hexanes). IR cm-1 (neat): 3796, 3667, 3360, 3250, 2922, 1715, 1648, 1601,
1516, 1343, 1258, 1232, 1095, 1061, 978, 843, 799, 753, 702. 1H-NMR d
(500 MHz, CDCl3): 8.32 (d, J 8.5, 2H), 7.83 (d, J 15.5, 1H), 7.74 (d, J 8.5, 2H), 6.78
(d, J 16, 1H), 6.64–6.47 (m, 5H), 4.94 (dd, J 3.0 and 8.0, 1H), 3.93–3.88 (m, 1H),
3.50–3.45 (m, 1H). MS (direct probe: ES ?): calc. mass = 312, actual mass = 313
(M ? H).
Compound 3e
Orange-yellow solid; yield = 68%; MP = 135–138°C; Rf = 0.4 (2% CH3OH/
CHCl3). IR cm-1 (neat): 3342, 3253, 2906, 1649, 1559, 1545, 1512, 1253, 1173,
1088, 1023, 824, 528.; 1H-NMR d (500 MHz, CDCl3): 7.60 (d, 15.0, 1H), 7.45 (dd,
J 2.0, 6.5, 2H), 7.19 (dd, J 2.0, 8.0, 2H), 6.90 (dd, J 2.0 and 6.5, 2H), 6.26 (d, J 15.5,
1H), 6.19 (dd, J 2.0, 6.0, 2H), 4.80 (dd, J 3.5, 8.0, 1H), 3.84 (s, 3H), 3.82–3.77 (m,
1H), 3.48–3.43 (m, 1H). MS (direct probe: ES?): calc. mass = 312, actual
mass = 295 (M ? H - H2O), 351 (M ? K).
Compound 3g
Yellow solid; yield = 50%; MP = 125°C (decomp); Rf = 0.6 (2% CH3OH/
CHCl3). IR cm-1 (mixture): 3270, 3049, 1659, 1600, 1515, 1410, 1342, 1244,
1
1171, 1108, 978, 841, 750. H-NMR d (500 MHz, DMSO) (mixture-double and
single bound): 8.29 (dd, J 2.0, 8.0, 2H), 7.88 (d, 8.0, 2H), 7.68 (dd, 2.5, 12.0, 2H),
7.52 (d, 16.0, 1H), 7.34 (d, 8.0, 2H), 6.92 (d, 16.0, 1H), 5.53 (d, 6.0, 1H), 4.65 (m,
1H), 3.50–3.40 (m, 1H). MS (direct probe: ES?): calc. mass = 327, actual
mass = 310 (M ? H - H2O).
Method C: for preparation of 3b
The nitro compound was reduced to an amine as described under Method C. The
amine was combined with PyBOP (1.5 eq) and carboxylic acid (1.5 eq). The

Med Chem Res (2010) 19:1141–1152
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mixture was suspended in dry methylene chloride and DIPEA (3 eq) was
introduced. The resulting clear reaction mixture was stirred at room temperature
for 4 days. The solvent was removed under reduced pressure and the product was
redissolved in CHCl3 and washed twice with water. The organic layer was dried
over sodium sulfate, then filtered, and the solvent was removed under reduced
pressure to obtain the crude product. The product was columned through silica gel
in 0–10% CH3OH/CHCl3 to get the desired compound.
Compound 3b
White solid; yield = 36%; MP = 170–172°C; Rf = 0.3 (2.5% CH3OH/CHCl3). IR
cm-1 (neat): 3349, 3266, 3080, 2934, 1647, 1594, 1507, 1455, 1414, 1341, 1295,
1
1250, 1170, 1097, 1061, 1025, 988, 831, 767, 704, 529. H-NMR d (500 MHz,
CDCl3): 7.85 (t, J 2.5, 1H), 7.52 (d, J 15.5, 1H), 7.38–7.35 (m, 3H), 7.26 (t, J 6.5,
2H), 7.18 (t, J 7, 1H), 6.80 (d, J 8.5, 2H), 6.37 (d, J 16.0, 1H), 4.81 (d, J 8.5, 1H),
3.83 (m, 1H), 3.77 (s, 3H), 3.31–3.26 (m, 1H). MS (direct probe: ES?): calc.
mass = 297, actual mass = 298 (M ? H).
Method D: for preparation of 3f
The nitro compound was reduced to an amine as described under Method C. The
amine was dissolved in dry DMF (3 ml). To this amine solution was added
carboxylic acid (1.1 eq), EDCI (2 eq), and DIPEA (0.1 eq), and the reaction
mixture was stirred at room temperature overnight. The solvent was removed under
reduced pressure and the residue obtained was dissolved in CHCl3 and washed with
water. The organic layer was dried over sodium sulfate, then filtered, the solvent
was removed, and the residue was column purified over silica gel using methanol
and chloroform.
Compound 3f
Off-white solid; yield = 20%; MP = 140°C; Rf = 0.4 (10% CH3OH/CHCl3). IR
cm-1 (neat): 3897, 3797, 3667, 3644, 3358, 2928, 2856, 2769, 1776, 1733, 1695,
1565, 1438, 1394, 1341, 1306, 1283, 1251, 1208, 1173, 1097, 1042, 841, 800, 755,
1
730, 683, 562. H-NMR d (500 MHz, CDCl3): 9.00 (s, 2H), 7.32 (dd, J 4.0, 9.0,
1H), 7.21 (d, J 8.5, 1H), 7.05 (d, J 2.0, 1H), 6.98 (dd, J 2.5, 8.5, 1H), 6.74 (d, J 2.0,
2H), 6.70 (dd, J 2.0, 6.5, 2H), 4.85 (dd, J 2.0, 8.5, 1H), 3.89 (m, 1H), 3.86 (s, 3H),
3.57–3.51 (m, 1H). MS (direct probe: ES?): calc. mass = 325, actual mass = 308
(M ? H - H2O).
Cell studies
B16 murine melanoma and L1210 murine leukemia cell lines were obtained from
the American Type Tissue Culture Collection (ATCC). Cells were grown in
Dulbecco’s Modified Eagle Medium (DMEM; Atlanta Biologicals) with 10% fetal
bovine serum, 2 mM Hepes buffer (Mediatech Cellgro, 25-060-Cl), 2 mM

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Med Chem Res (2010) 19:1141–1152
L-glutamine (Mediatech Cellgro), and 2 mM penicillin/streptomycin (50,000 U
penicillin/50,000 lg streptomycin; Atlanta Biologicals).
Cytotoxicity studies utilizing the MTT assay
The compound stock solution was prepared by dissolving the sample in DMSO to
obtain a 1.75 9 10-2 M solution. Serial 1:10 dilutions were done in DMSO by
transferring 10 ll of the stock solution into 100 ll of DMSO. From the solutions,
10 ll was then transferred into another well containing 100 ll of DMSO. This was
repeated to prepare solutions of concentrations 1.75 9 10-3–1.75 9 10-10 M.
These stock solutions were then further diluted with DMEM (24 ll stock solution in
176 ll medium). The addition of these drug stock solutions (5 ll) to wells
containing 100 ll medium per cell suspension resulted in final drug concentrations
ranging from 1.0 9 10-12–1.0 9 10-4 M.
This cell suspension (100 ll) was added to a 96-well flat-bottom cell culture
plate. At this concentration, 8000 (L1210) and 4000 (B16) cells were seeded in each
well. Drug solutions diluted in DMSO and DMEM as explained below were added
to each well (5 ll/well). Quadruplicate wells were prepared for each drug
concentration. Plates were incubated for 72 h at 37°C in a 5% CO2 humidified
atmosphere. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-biphenyl tetrazolium bromide)
was dissolved in PBS (5 mg/ml). After the indicated cell incubation period, 10 ll of
stock MTT solution was added to each well and the plates were further incubated for
4 hundred incubation conditions. After this incubation, 100 ll acid isopropanol
solution (16 ll 12.1 N HCl in 5 ml isopropanol) was added to each well. The
contents of the well were mixed thoroughly by pipetting the cell suspension up and
down, and the plates were allowed to sit at room temperature for 15 min to allow for
full development of the purple color. Plates were then read on a Dynatech Plate
Reader, utilizing Dynex Revelation 3.2 software, with a test wavelength of 570 nm
and a reference wavelength of 630 nm. The dose inhibiting the growth by 50%
(IC50) was extrapolated from curves generated based on the averages of the
absorbance data (4 points/concentration).
Flow cytometry experiment: propidium iodide (PI) staining
A stock solution of 3f was made in DMSO so that the addition of 5 ll of the solution
into 5 ml of medium would result in the desired concentrations. Cells were counted
and resuspended at a concentration of 1.5 9 105 cells/ml, and 5 ml of this cell
suspension was pipetted into a small culture flask. The drug solutions were added
(5 ll) and the cells were incubated at 37°C and 5% CO2 for 24 h. Instead of fixing
the cells as done in previous studies in this lab, cells were transferred to sterile tubes
(15 ml) via pipette and set on ice. Shortly prior to analysis, the cells were harvested
via centrifugation (1000 rpm, 10 min) and the PI solution was added to the cells.
Approximately 10 min before the desired time of analysis, cells were stained with
1 ml PI. The PI stock solution was prepared by dissolving 0.8 mg of PI in 0.8 ml of
DI H2O. The PI staining solution was then prepared by the addition of 0.6 ml of the
PI stock solution to 12 ml of sample buffer. RNase A (EC 3.2.27.5; Sigma) was then

Med Chem Res (2010) 19:1141–1152
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added (1200 Kunitz units; *17 mg. This solution was then thoroughly mixed
before its (1 ml) addition to the cells, which were pelleted by centrifugation at
3000 rpm for 5 min at 4°C. Cells were kept on ice until they were analyzed by flow
cytometry. The analysis was completed on a Becton–Dickinson FACScan.
Flow cytometry studies: annexin V and PI costaining experiment
The same treatments were prepared and incubated for 3 h at 37°C and 5% CO2.
According to instructions (Sigma annexin V FITC kit), the cells were transferred to
15-ml tubes, harvested via centrifugation (2000 rpm, 5 min), and washed twice with
sample buffer (0.5 g glucose in 500 ml Ca2?/Mg2?-free PBS; Electron Microscopy
Sciences, Fort Washington, PA, USA). One ml 1 9 buffer was added to the cells
and the cells were transferred to 5-ml round-bottom tubes (Fisher), which were kept
on ice until staining. The buffer was prepared by diluting 1 ml of 10 9 buffer
(Sigma) up to 10 ml with DI H2O. Ten minutes before analysis 5 ll of annexin V
and 10 ll of PI were added to each sample. Three standards were used to calibrate
the FACScan: unstained, annexin V only, and PI only. A sample of costained cells
was also prepared as a standard. Cisplatin was used as a positive control. The results
for the double-stained analyses were displayed as dot plots.
Results and discussion
Chemistry
The synthesis of the 1-phenylethanolamine carboxamide derivatives follows a direct
strategy as shown in Scheme 1. The reaction of substituted benzaldehydes with
nitroethane under the Henry aldol condensation conditions yielded the nitropheny-
lethanol derivatives 2a and 2b (Desai et al., 2004; Kleina et al., 2002). The nitro
groups of these compounds were reduced by catalytic hydrogenation in the presence
of 5% Pd/C and the resulting amino compounds were reacted with various carboxylic
acids under different conditions to give the 1-phenylethanolamine arylcarboxamides
3a–3g in reasonable yields (Scheme 1). The compounds were purified by silica gel
column chromatography using methanol/chloroform or ethyl acetate/hexane solvent
systems. The compounds were characterized using a combination of techniques,
1
including FT-IR, 500-MHz H-NMR, and mass spectroscopy (MS).
In vitro studies on B16 and L1210 cells
The cytotoxic activity of compounds 3a–3g were studied against the growth of
murine B16 and L1210 cells in culture and using the MTT assay (Sato et al., 2005;
Kupchinsky et al., 2004; Carmichael et al., 1987). Cells were treated continuously
for 3 days and their cytotoxicity was compared to that of untreated cells. The
cytotoxicity results, expressed as IC50 values, are reported in Table 1. These results
indicate that the compounds were generally slightly more active against leukemia
over melanoma cells. The 40-amino compounds were generally more active among

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Med Chem Res (2010) 19:1141–1152
O
OH
Ar1
H
OH
Method A
Method B or C or D
N
Ar2
NO2
H
CH3NO2
Ar1
Ar1
O
3a, Ar1 = Ph, Ar2= 3-MeO-Ph-CH=CH
3b, Ar1 = Ph, Ar2 = 4-MeO-PhCH=CH
3c, Ar1 = Ph, Ar2 = 5-MeO-Indole-2-
3d, Ar1 = Ph, Ar2= 4-NO2-Ph-CH=CH
3e, Ar1=4-NH2-Ph, Ar2=4-MeO-Ph-CH=CH
3f, Ar1=4-NH2-Ph, Ar2=5-MeO-Indole-2-
3g, Ar1=4-NH2-Ph, Ar2=4-NO2-Ph-CH=CH
2a, Ar1=Ph,
2b, Ar1=4-(NO2)Ph
Scheme 1 Synthesis of the target compounds 3a–3g. Method A: CH3CN, K3PO4, room temperature (rt),
overnight. Method B: aH2, Pd–C, CH3OH; bacid chloride, triethylamine, 0°C. Method C: aH2, Pd–C,
CH3OH; PyBOP, carboxylic acid, dry CH2Cl2, DIPEA, rt, 4 days. Method D: aH2, Pd–C, CH3OH;
carboxylic acid, EDCI, DIPEA, dry DMF, rt, overnight
the compounds tested, with 5-methoxyindole derivatives 3c and 3f being more
active than their cinnamoyl congeners. The IC50 values for compounds 3c and 3f fell
in the range of 25–87 lM. This finding is noteworthy because indoles were not
included in the earlier report as studied by Nesterenko’s group (2003). For reasons
that are currently unclear, indoles apparently confer additional cytotoxic properties
to this class of 1-phenylethanolamines.
Flow cytometry studies
Based on the cytotoxic activity against the growth of L1210 and B16 cells in
culture, as well as the increased polarity of compound 3f over 3c, the 5-
methoxyindole compound 3f was selected for flow cytometry studies for the purpose
of probing its apoptosis-inducing properties (Sato et al., 2005; Kupchinsky et al.,
2004). The first test utilized PI staining, which detects living versus dead cells, and
those cells that are most likely going through the dying process. Specifically, the M1
area of the histogram given in Fig. 2 displays healthy living cells, M2 shows cells
undergoing change (dying), and M3 displays those that have already died. In these
studies the L1210 cells were treated for 3 h at 10 times the IC50 concentration.
Figure 2a depicts the control untreated cells, and all cells were alive (M1). On the
other hand, cells that were treated with compound 3f (Fig. 2b) demonstrated a
significant number that were dying (M2) or dead (M3). The results for compound
3f-treated cells were remarkably similar to those for cells treated with cisplatin (10
times IC50 concentration also; Fig. 2c), which is known to induce cancer cells to
undergo apoptosis (Sato et al., 2005; Kupchinsky et al., 2004; Ormerod et al.,
1994). The reported IC50 for cisplatin against L1210 cells in culture is 100 lM (Sato
et al., 2005; Kupchinsky et al., 2004).
Although the PI study gave encouraging results, the results were not equivocal
with regard to apoptosis induction, because it was difficult to discern cells that died
from apoptosis versus necrosis, as cells in M3 could come from either pathway. A
more definitive analysis was required to more accurately understand the action of
the compound, and in this case a costain method was used. This method employed

Med Chem Res (2010) 19:1141–1152
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Med Chem Res (2010) 19:1141–1152
Fig. 2 Histograms from fluorescence-activated cell sorting (FACS) studies run on compound 3f,
cisplatin, and a control. L1210 murine leukemia cells were treated for 3 h at 10 times their IC50 values
and stained with PI. a Cell control; b compound 3f (500 lM); c cisplatin (1000 lM)
Fig. 3 Dot plots show the results of FACS studies on L1210 cells treated for 3 h at 10 times their IC50
values and stained with annexin V and PI costain. a Cells treated with compound 3f, b control untreated
cells, and c cells treated with 10 times the IC50 of cisplatin
PI staining, which detects changes in DNA structure, and a costain, annexin V,
which detects membrane breakdown by staining phosphatidylserine movement from
the cytoplasm to the cell surface during apoptosis. When combined with the PI stain,
the dot plot accurately detects those cells which are in the process of undergoing
apoptosis (Li et al., 2008) (Sigma–Aldrich annexin V-FITC Apoptosis Detection
Kit).
The results of the costain experiment (Fig. 3) show a significant effect of
increasing apoptosis in L1210 cells that were treated with compound 3f. Cells were
treated for 3 h at 10 times the IC50 concentration of compound 3f or cisplatin. In
Fig. 3, R1 shows the percentage of dead cells, R2 shows living cells, and R3 shows
cells undergoing apoptosis. For compound 3f (Fig. 3a), 48% of the cells were alive,
7% were dead, and 41% were undergoing apoptosis. In comparison, of the control
untreated cells (Fig. 3b), 32% of the cells were alive, 5.3% were dead, and 30%
were undergoing apoptosis. On the other hand, cisplatin-treated cells (Fig. 3c)
showed 36% living, 7% dead, and 34% undergoing apoptosis. These results further
supported the results from the PI study, in which the nonmethyl 40-amino-1-
phenylethanolamine 2-indolecarboxamide 3f was able to induce cancer cells to
undergo apoptosis.

Med Chem Res (2010) 19:1141–1152
1151
Conclusion
Seven novel analogues of 1-phenylethanolamine carboxamide derivatives were
tested and found to be active against the growth of B16 and L1210 murine cancer
cells in culture. Flow cytometry studies on a selected compound, 3f, provided
evidence that these compounds are capable of promoting L1210 cells to undergo
apoptosis, compared to untreated controls and cells treated with a cisplatin. Further
structural modification of compounds 3c and 3f is an attractive next step in this
project.
Acknowledgments The authors are grateful for the NIH INBRE grant that support of this work. The
authors also thank the Greenville Hospital System—Oncology Research Institute for carrying out the flow
cytometry experiments.
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