Synthesis and cytotoxicity of substituted ethyl 2-phenacyl-3-phenylpyrrole-4-carboxylates

Article abstract of DOI:10.1002/ardp.200390018

The substituted ethyl-2-phenacyl-3-phenylpyrrole-4-carboxylates were synthesized by a condensation of a beta-chloroenal and an alpha-aminoketone under neutral conditions. They proved to be potent cytotoxic agents against the growth of murine L1210 and P38

Full text of DOI:10.1002/ardp.200390018

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 181–190
Ethyl 2-Phenacyl-3-phenylpyrrole-4-carboxylates 181
Michael A. Evansa,
Synthesis and Cytotoxicity of Substituted Ethyl
2-Phenacyl-3-phenylpyrrole-4-carboxylates
Daniel C. Smitha,
Justin M. Holuba,
Anthony Argentia,
Mafoloe Hoffa,
The substituted ethyl-2-phenacyl-3-phenylpyrrole-4-carboxylates were synthesized
by a condensation of a beta-chloroenal and an alpha-aminoketone under neutral
conditions.They proved to be potent cytotoxic agents against the growth of murine
L1210 and P388 leukemias and human HL-60 promyelocytic leukemia, HuT-78 lym-
phoma, and HeLa-S3 uterine carcinoma. Selective compounds were active against
the growth of Tmolt3 and Tmolt4 leukemias and THP-1 acute monocytic leukemia,
liver Hepe-2, ovary 1-A9, ileum HCT-8 adenocarcinoma, and osteosarcoma HSO.A
mode of action study in HL-60 cells demonstrated that DNA and protein syntheses
were inhibited after 60 min at 100 µM. DNA and RNA polymerases, PRPP-amido
transferase, dihydrofolate reductase, thymidylate synthase, and TMP kinase activi-
ties were interfered with by the agent with reduction of d[NTP] pools.Nonspecific in-
teraction with the bases of DNA and cross-linking of the DNA may play a role in the
mode of action of these carboxylates.
Gerard A. Dalglisha,
Donna L. Wilsona,
Brett M. Taylora,
Joshua D. Berkowitza,
Bruce S. Burnhama,
Keith Krumpeb,
John T. Guptonc,
Tanya C. Scarlettd,
Richard W. Durham, Jrd,
Iris H. Halld
a
Department of Chemistry
and Biochemistry,
Rider University,
Lawrenceville, NJ 08648,
USA
Keywords: Antitumor; Pyrroles, Cytotoxicity
Received: July 29, 2002 [FP717]
b
Department of Chemistry,
UNC-Asheville, Asheville,
NC 28801, USA
Department of Chemistry,
c
University of Richmond,
Richmond, VA 23173, USA
Division of Medicinal
d
Chemistry and Natural
Products,
School of Pharmacy,
University of North Carolina,
Chapel Hill NC 27599-7360,
USA
against the growth of human HL-60 cell leukemia sup-
pressing the DNA and RNA syntheses but not protein
Introduction
Previously it has been shown that alkyl-3,4-bis(4-meth-
oxyphenyl) pyrrole-2-carboxylates [1] and 2,3,4-trisub-
stituted pyrroles [2] demonstrated potent cytotoxicity
against the growth of murine and human tumors, i.e.
leukemia, lymphomas, Tmolt4 leukemia, THP-1 acute
monocytic leukemia, and HeLa-S3 uterine carcinoma. In
addition, it also has been documented that 2,4-disubsti-
tuted and 2,3,4-trisubstituted brominated pyrroles [3]
successfully demonstrated potent cytotoxicity against
the growth of solid human tumors.These compounds af-
forded ED50 values of Յ 4 µg/mL, which is required for
significant activity. All of the compounds were active
synthesis in 60 min at 100 µM. Mode of action studies
revealed the compounds suppressed DNA polymerase
α, m-RNA polymerase, t-RNA polymerase, dihydro-
folate reductase, and nucleoside kinase activities. In
calf-thymus DNA studies, certain compounds caused
alkylation of the bases of DNA. DNA fragmentation oc-
curred after 24 h with certain compounds, while other
compounds appeared to cause cross-linking of the DNA
stands [3].
Results
Correspondence: Iris H. Hall, Division of Medicinal Chemistry
and Natural Products, School of Pharmacy, University of
North Carolina, Chapel Hill NC 27599-7360, USA.
Phone: +1 919 962 0064, fax: +1 919 966 0204, e-mail:
iris_hall@unc.edu.
The substituted ethyl 2-phenacyl-3-phenylpyrrole-4-
carboxylates demonstrated potent activity against the
growth of murine L1210 lymphoid leukemia and P388
lymphocytic leukemias [Table 1].ED50 values of less than
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0365-6233/03/0181 $ 17.50+.50/0

182 Evans et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 181–190
Table 1. Cytotoxicity [ED50 values = µg/mL] of carboxylates in murine and human tumors.
Tumor cell line
9 c
9 b
9 d
9 a
6-MP
Ara-C
VP-16 Mitomy- 5-FU
Etopo- cin C
side
L1210 mouse leukemia
P388 mouse lymphocytic
leukemia
3.09
2.77
3.09
3.42
3.59
2.11
2.64
3.38
2.43
2.04
3.07
0.79
1.83
0.99
1.41
1.41
HL-60 human leukemia
Tmolt3 T cell leukemia
T molt4 T cell leukemia
HuT-78 lymphoma
THP-1 Acute monocytic
leukemia
2.22
4.70
5.88
3.22
4.06
1.44
6.28
5.72
2.13
4.57
3.79
3.22
3.40
2.75
3.56
1.79
4.17
3.20
2.96
2.63
3.35
1.62
2.67
1.68
3.03
4.00
2.67
2.36
2.50
2.54
4.43
1.00
1.92
1.33
3.27
5.28
2.14
2.75
5.81
1.12
HeLa-S3 uterine
KB nasopharynx
Lung A-549
3.98
4.08
5.15
3.14
2.32
8.18
5.63
3.23
8.52
2.35
9.10
7.84
2.48
5.74
6.22
3.24
2.58
7.77
8.69
3.28
6.65
4.21
6.00
6.12
2.59
4.06
6.04
4.23
6.50
4.53
6.87
1.41
6.28
2.14
2.32
7.34
2.64
5.06
4.00
3.26
4.90
3.28
2.47
4.42
10.9
9.57
6.89
8.73
2.12
11.04
4.71
2.13
2.84
5.62
1.69
3.32
4.74
2.47
1.25
3.58
1.70
0.14
Liver Hepe-2
Ovary 1-A9
Breast MCF-7
Glioma UM 86
Ileum HCT-8
Prostate PL
Osteosarcoma HSO
Melanoma SK2
Normal RMPI 1788
6.64
8.84
4.46
1.15
5.39
12.45
1.88
6.24
11.00
2.44
1.13
2.80
3.57
3.53
–
0.45
1.60
0.15
1.60
6.82
1.28
1.30
2.54
9.13
6.86
–
0.86
10.53
–
8.73
5.93
4 µg/mL were considered significant according to the
NCI protocol [7].This was the concentration of test com-
pound required to cause 50 % tumor cell death after 3
days incubation. All four compounds were active against
the growth of human HL-60 leukemia, HuT-8 lymphoma,
and suspended HeLa-S3 uterine growth.Compounds 9 a
and 9 d were active against Tmolt4 T cell leukemia, and
THP-1 acute monocytic leukemia growth but only com-
pound 9 d was active against Tmolt3 T cell leukemia
growth. None of the compounds were active against the
growth of the solid tumors:KB nasopharynx, lung A-549,
prostate-PL, or human normal fibroblasts 1788.Yet, ile-
um adenocarcinoma HCT-8 growth was suppressed by
compounds 9 b, 9 c, and 9 d, osteosarcoma HSO by 9 c
and 9 d, liver-Hepe-2 carcinoma by compounds 9 a, 9 b,
and 9 c, ovary A-1carcinoma by compounds 9 b and 9 c.
Compound 9 d reduced the growth SK-2 melanoma and
compound 9 a reduced the growth of breast MCK-7 and
glioma UM-86.
DNA polymerase α activity was reduced 51 %, m-RNA
polymerase activity 47 %, r-RNA polymerase activity
39 % and t-RNA polymerase activity 50 % after 60 min at
100 µM. De novo purine synthesis was suppressed
23 % with the regulatory enzyme of the pathway, PRPP-
amido transferase, being inhibited 88 %. Dihydrofolate
reductase activity was reduced 93 %, thymidylate syn-
thase activity 26 % andTMP kinase activity 32 %.The de
novo pyrimidine pathway and its regulatory enzyme ac-
tivities were not affected by compound 9 a as was true for
IMP dehydrogenase, thymidine kinase, and TDP kinase
activities. The inhibition of the above enzyme activities
was reflected in the deoxyribonucleotide pool levels in
that dATP was reduced 24 %, dGTP, 19 %, dCTP 13 %,
and TTP 17 % after 60 min at 100 µM. ct-DNA studies
showed that compound 9 a caused a slight hyperchomic
shift in the UV absorption from 220–340 mm but there
was no change in the TM values for DNA denaturation or
for DNA viscosity after 24 h incubation at 100 µM.HL-60
leukemia cells after 24 h incubation at 100 µM demon-
strated evidence of cross-linking of the DNA molecules
[Fig. 4]. Compound 9 a did not inhibit DNA topoisomer-
ase I or II activities or elevate caspase 1 or 3 cleavage in
HL-60 cells at 100 µM.
A mode of action study in HL-60 leukemia cells with com-
pound 9 a showed that after 60 min at 100 µM, DNA
synthesis was suppressed 37 % and protein synthesis
52 % but there was no effect on RNA synthesis [Table 2].

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 181–190
Ethyl 2-Phenacyl-3-phenylpyrrole-4-carboxylates 183
Table 2. Effects of compound 9 a on Hl-60 leukemia cell metabolism after 60 min incubation.
Percent of control (X S.D.).
Assay (N = 6)
Control
25 µM
50 µM
100 µM
63 4*
DNA synthesis
RNA synthesis
100 6a
100 5b
100 6c
100 5d
100 4e
100 6f
100 6g
100 6h
100 6i
100 6j
100 7k
100 6l
100 6m
100 7n
100 6o
100 5p
100 4q
100 4r
100 6s
100 4t
100 6u
100 6v
100 6w
79 5*
72 4*
106
6
52 6*
43 4*
105
5
100
4
Protein synthesis
DNA polymerase α
mRNA polymerase
rRNA polymerase
tRNA polymerase
Ribonucleotide reductase
De novo purine synthesis
PRPP amido transferase
IMP dehydrogenase
De novo pyrimidine synthesis
Carbamyl phosphate synthetase
Aspartate transcarbamylase
Thymidylate synthase
Thymidine kinase
TMP kinase
49 5*
41 4*
58 4*
72 4*
48 4*
39 3*
53 5*
61 3*
50 4*
87
5
78 4*
89
101
100
5
4
5
87
98
86
6
5
3
95
4
77 3*
12 2*
60 4*
18 3*
105
93
97
5
4
5
96
91
93
103
89
133
95
3
3
4
7
5
7
4
5
91
88
90
86
4
3
4
4
128 6*
100
109
141
113
5
6
6
6
74 4*
132
5
68 4*
TDP kinase
Dihydrofolate reductase
d(ATP)
d(GTP)
d(CTP)
98
95
7
5
3*
29 6*
13 4*
76 + 5*
81 4*
87
5
d(TTP)
83 6*
* P Յ 0.001;
a
b
c
d
e
f
k
l
p
q
r
u
45011 dpm
4226 dpm
5343 dpm
7125 dpm
5693 dpm
8394 dpm
5151 dpm
63565 dpm
17646 dpm
0.164 OD units
4658 dpm
7316 dpm
1.242 µmoles citrulline
1.030 mol N-carbamyl aspartate
13890 dpm
1511 dpm
320 dpm
286 dpm
0.092 OD units
9.02 pmoles
11.21 pmoles
13.65 pmoles
16.73 pmoles
g
h
i
v
m
n
w
s
t
j
o
purine synthesis at its regulatory enzyme, PRPP-amido
transferase, but the de novo pyrimidine pathway ap-
peared not to be affected after 60 min. The marked re-
duction in dihydrofolate reductase activity afforded by
the compound would also reduce one-carbon transfer for
de novo purine synthesis.Nucleoside kinase activity was
marginally reduced as was the d[NTP] pools after
60 min. These pool levels are the net of two metabolic
events in that the d[NTP] pools should rise due to the in-
hibition of DNA polymerase activity which would not al-
low the incorporation of d[NTP] into the new strand of
DNA whereas inhibition of purine synthesis would re-
duce the available d[NTP]s for the incorporation.The ob-
servation that RNA synthesis was not affected signifi-
cantly by the agent in 60 min is not unusual in that RNA
Discussion
The substituted ethyl 2-phenacyl-3-phenylpyrrole-4-car-
boxylates demonstrated good cytotoxic activity against
murine and human cancers similar to previously report-
ed related compounds in that they were more active
against suspended tumor cells and more selective to-
wards inhibiting cell growth of cells derived form solid tu-
mors.It should be noted that no marked structure activity
relationship existed for the cytotoxicity of the pyrrole de-
rivatives tested. The suppression of DNA synthesis in
HL-60 leukemia cells by the pyrrole derivative was due to
multiple enzymes being targeted by the drug. DNA tem-
plate utilization was affected since all of the polymerase
activities were reduced in a concentration manner. The
agent also seemed to be directed towards de novo

184 Evans et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 181–190
to DNA ratios in mammalian cells is 10:1 and the block-
age of purine synthesis would affect DNA synthesis ear-
lier than RNA synthesis.The DNA molecule itself can not
be eliminated as a target of the agent in that a slight hy-
perchromic shift occurred. This binding of the drug to
DNA is probably not by alkylation of the nucleoside bas-
es by the pyrrole ring but rather it probably is a nonspecif-
ic interaction between the drug and the chromatin mate-
rail.The drug did not cause intercalation between nucle-
oside base pairs and no DNA fragmentation occurred in
24 h.This is consistent with the agent having no effect on
DNA topoisomerase I and II activities or the repair DNA β
polymerase activity.The agent did not appear to function
in a manner so that it caused tumor cell death due to acti-
vation of apoptosis since the caspase proteolytic en-
zymes were not activated. HL-60 cells did demonstrate
cross-linking of the DNA-strands after 24 h incubation
suggesting the possibility of a nonspecific interaction
with chromatin structure.
Figure 2. Preparation of the salt of the α-aminoketone
from a 2-bromoacetophenone.
Compared to the previous pyrrole derivatives, the cur-
rent pyrrole demonstrated more potent inhibition of pro-
tein synthesis and dihydrofolate reductase activity but
was similar in its reduction of PRPP-amido transferase
and TMP kinase activities and cross-linking of the DNA
strands [1–3].However, the current pyrrole did not cause
inhibition of IMP dehydrogenase or ribonucleotide
reductase activities as observed with other pyrroles
[1–3].
Figure 3. Synthesis of the 2,3,4-trisubstituted pyrroles.
Chemistry
The 2,3,4-trisubstituted pyrroles (9 a–9 d) were pre-
pared using methods previously reported [1–5] by heat-
ing an α-aminoketone (7 or 8) and a β-chloroenal
(4 a–4 d) in DMF under neutral conditions. In this [3+2]-
cycloaddition reaction, the nitrogen and C-1 are from the
aminoketone and the remaining three carbons are pro-
vided from the β-chloroenal. The chloroenal was pre-
pared from an acetophenone in three steps. A Claisen-
type condensation [6] of the acetophenone (1 a–1 d) and
diethyl carbonate is carried out in toluene and sodium
Figure 4. HL-60 DNA strand scission after 24 h at
100 µM.
hydride, which produces a β-ketoester (2 a–2 d) in good
yield.The ketoester is treated with DMF-acetal quantita-
tively yielding a vinylogous amide (3 a–3 d), which is
then refluxed in POCl3, and CH2Cl2 for 2–3 h followed by
hydrolysis in THF/H2O affording the β-chloroenal
(4 a–4 d) yields of 14–34 % (Figure 1).The pTSA salt of
Figure 1. Preparation of the β-chloroenal from an ace-
tophenone. R = 3-Cl (a), 4-Cl (b), 4-Br (c), 4-CH3 (d).

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 181–190
Ethyl 2-Phenacyl-3-phenylpyrrole-4-carboxylates 185
gen atmosphere.The mineral oil was removed by washing with
2× 50 mL hexanes.To the NaH was added 400 mL toluene and
diethyl carbonate (47 mL, 45.8 g, 0.389 mol, 5.1 Eq) with stir-
ring. A solution of 4Ј-chloroacetophenone (10 mL, 11.9 g,
0.077 mol) in 10 mL toluene was added dropwise to the flask
containing the NaH and diethyl carbonate. The mixture was
stirred at reflux under N2 (g) for 18 h.After cooling to RT, 25 mL
of glacial acetic acid was added to the stirring mixture.The mix-
ture was then poured over ice (500 mL) and conc.HCl (10 mL).
The aqueous layer was washed with 3× 50 mL ethyl acetate.
The combined organic layers were washed with 3× 50 mL cold
H2O, 3× 50 mL sat.NaHCO3 (aq) and 3× 50 mL brine.The solu-
tion was dried over anhydrous MgSO4, filtered and the solvent
was removed under reduced pressure.The crude oil was puri-
fied by Kugelrohr distillation, bp = 121–123 °C at 0.5 mm Hg,
affording 9.49 g (.0419 mol) of a pale yellow oil in a 54 % yield.
TLC showed a single spot with an Rf = 0.53 in 7:3 hexanes:ethyl
acetate. IR (neat on NaCl): ν = 1742 cm–1 (C=O), 1684 cm–1
(C=O), 1607 cm–1 (aromatic C-C). 1H NMR of keto tautomer
(CDCl3): δ = 7.81 (d, 2 H, J = 8.2 Hz), 7.37 (d, 2 H, J = 8.2 Hz),
4.13 (q, 2 H, J = 7.2 Hz), 3.88 (s, 2 H), 1.18 (t, 3 H, J = 7.2 Hz)
ppm. 13C NMR (CDCl3): δ = 192, 168, 141, 132, 130.3, 129.5,
62, 46, 15 ppm. (C11H11ClO3).
an α-amino ketone (7) is conveniently prepared in two
steps [7] by first forming an α-azidoketone (6) from the 2-
bromoacetophenone (5) and NaN3 in DMSO followed by
a reduction carried out by Ph3P and pTSA (Figure 2).Fi-
nally, heating the β-chloroenal and the salt of the α-ami-
noketone in dry DMF forms the 2,3,4-trisubstituted pyr-
role (9 a–9 d, Figure 3).
Acknowledgements
The authors would like to thank American Cyanamid for
its Grant-In-Aid to BSB, Project SEED of the American
Chemical Society and the NSF NMR-Collaborative
Training Partnership Grant (DUE-9952369) for the 300
MHz Bruker Avance FT-NMR at Rider University.
Experimental
Materials and methods
All chemicals and reagents were obtained from Aldrich Chemi-
cal Company (Milwaukee, WI) and used as received except for
dry solvents, which were dried and distilled using standard
procedures [8]. TLC was performed using silica gel 60F 254
plates (silica gel on plastic, Aldrich Chemical Company). Melt-
ing points were obtained on a Thomas-Hoover Uni-melt appa-
ratus (capillary method), and were uncorrected. IR spectra
were obtained on a Perkin-Elmer 1600 FTIR spectrometer on
sodium chloride plates or in a potassium chloride liquid cell in
CHCl3 or CDCl3. NMR spectra were obtained on a 300 MHz
Bruker Avance FT-NMR spectrometer using tetramethylsilane
as an external standard for 1H and 13C spectra (δ = 0 ppm).Ele-
mental analyses were performed by QuantitativeTechnologies,
Inc. (Whitehouse, NJ).
Preparation of ethyl 3-(4Ј-bromophenyl)-3-oxopropionate (2 c)
Sodium hydride (6.94 g, 0.174 mol, 2.6 Eq) as a 60 % disper-
sion in mineral oil was placed in a 1L 3-neck flask under nitro-
gen atmosphere. The mineral oil was removed by washing 2×
with 50 mL hexanes. To the NaH was added 300 mL toluene
and diethyl carbonate (48 mL, 46.8 g, 0.396 mol, 5.9 Eq) with
stirring.
A solution of 4Ј-bromoacetophenone (13.29 g,
0.0668 mol) in 100 mL toluene was added dropwise to the
flask containing the NaH and diethyl carbonate. The mixture
was stirred at reflux under N2 (g) for 18 h. After cooling to RT,
30 mL of glacial acetic acid was added to the stirring mixture.
The mixture was then poured over ice (500 mL) and conc. HCl
(20 mL). The aqueous layer was washed 2× with 50 mL ethyl
acetate. The combined organic layers were washed with 3×
50 mL cold H2O, 3× 50 mL sat. NaHCO3 (aq), and 3× 50 mL
brine. The solution was dried over anhydrous MgSO4, filtered,
and the solvent was removed under reduced pressure. The
crude oil was purified by Kugelrohr distillation, bp = 129–131 °C
at 0.6 mm Hg, affording 14.85 g (0.05477 mol) of a dark brown
oil in a 82 % yield.TLC showed a single spot with an Rf = 0.59 in
7:3 hexanes:ethyl acetate. IR (neat on NaCl): ν = 1688 cm–1
(C=O), 1620 cm–1 (C=O), 1586 cm–1 (aromatic C-C). 1H NMR
of keto tautomer (CDCl3): δ = 7.76 (d, 2H, J = 8.5 Hz), 7.76 (d,
2 H, J = 8.5 Hz), 4.08 (q, 2 H, J = 7.2 Hz), 3.82 (s, 2 H), 1.13 (t,
3 H, J = 7.2 Hz) ppm.13C NMR (CDCl3):δ = 191, 167, 140, 135,
131, 129, 62, 46, 14 ppm. (C11H11BrO3).
Preparation of ethyl 3-(3Ј-chlorophenyl)-3-oxopropionate (2 a)
Sodium hydride (6.5 g, 0.16 mol, 2.5 Eq) as a 60 % dispersion
in mineral oil was placed in a 1L 3-neck flask under nitrogen at-
mosphere. The mineral oil was removed by washing 2× with
50 mL hexanes.To the NaH was added 150 mL toluene and di-
ethyl carbonate (38.21 g, 0.323 mol, 5 Eq) with stirring. 3Ј-
Chloroacetophenone (10.0 g, 0.0647 mol) was added drop-
wise to the flask containing the NaH and diethyl carbonate over
7 min.The mixture was stirred at reflux under N2 (g) for 12 h.Af-
ter cooling to RT, 25 mL of glacial acetic acid was added to the
stirring mixture.The mixture was then poured over ice (40 mL)
and conc.HCl (10 mL).The aqueous layer was washed 3× with
50 mL ethyl acetate. The combined organic layers were
washed 2× with 50 mL sat. NaHCO3 (aq) and 2 × 50 mL brine.
The solution was dried over anhydrous MgSO4, filtered, and the
solvent was removed under reduced pressure. The crude oil
was purified by Kugelrohr distillation, bp = 114–116 °C at
0.5 mm Hg, affording 6.96 g (0.031 mol) of a clear oil in a 47 %
yield. TLC showed a single spot with an Rf = 0.43 in 9:1 hex-
anes:ethyl acetate. IR (neat on NaCl): ν = 1744 cm–1 (C=O),
1693 cm–1 (C=O), 1628 cm–1 (C=O), 1594 cm–1 (aromatic
C-C). 1H NMR of keto tautomer (CDCl3): δ = 7.83 (s, 1H, 7.02–
7.75 (m, 3 H), 4.11 (q, 2 H, J = 7.2 Hz), 3.85 (s, 2 H), 1.22 (t, 3 H,
J = 7.2 Hz) ppm.13C NMR (CDCl3):δ = 192, 167, 138, 136, 134,
131, 129, 127, 62, 46, 14 ppm. (C11H11ClO3).
Preparation of ethyl 3-(4Ј-methylphenyl)-3-oxopropionate (2 d)
Sodium hydride (11.2 g, 0.28 mol, 2.5 Eq) as a 60 % disper-
sion in mineral oil was placed in a 1L 3-neck flask under nitro-
gen atmosphere. The mineral oil was removed by washing 2×
with 50 mL hexanes. To the NaH was added 400 mL toluene
and diethyl carbonate (27 mL, 0.22 mol, 2 Eq) with stirring.
A
solution of 4Ј-methylacetophenone (15.0 mL, 15.1 g,
0.112 mol) in 100 mL toluene was added dropwise to the flask
containing the NaH and diethyl carbonate. The mixture was
stirred at reflux under N2 (g) for 10 h.After cooling to RT, 60 mL
of glacial acetic acid was added to the stirring mixture.The mix-
ture was then poured over ice (500 mL) and conc.HCl (30 mL).
The aqueous layer was washed 3× with 50 mL CHCl3. The
combined organic layers were washed 3× with 50 mL cold H2O,
3× with 50 mL sat. NaHCO3 (aq) and 3× with 50 mL brine.The
solution was dried over anhydrous MgSO4, filtered and the
Preparation of ethyl 3-(4Ј-chlorophenyl)-3-oxopropionate (2 b)
Sodium hydride (6.78 g, 0.169 mol, 2.2 Eq) as a 60 % disper-
sion in mineral oil was placed in a 1L 3-neck flask under nitro-

186 Evans et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 181–190
Preparation of ethyl 3-(dimethylamino)-1-(4Ј-methylphenyl)-
prop-2-enone-2-carboxylate (3 d)
solvent was removed under reduced pressure. The crude oil
was purified by Kugelrohr distillation, bp
= 139 °C at
0.2 mm Hg, affording 13.06 g of a clear oil in a 58 % yield.TLC
showed a single spot with an Rf = 0.67 in 7:3 hexanes:ethyl ace-
tate. IR (neat on NaCl): ν = 1742 cm–1 (C=O), 1684 cm–1
(C=O), 1607 cm–1 (aromatic C-C). 1H NMR of keto tautomer
(CDCl3): δ = 7.81 (d, 2H, J = 8.1 Hz), 7.23 (d, 2 H, J = 8.1 Hz),
4.18 (q, 2 H, J = 7.2 Hz), 3.91 (s, 2 H), 2.38 (s, 3 H), 1.23 (t, 3 H,
J = 7.2 Hz) ppm.13C NMR (CDCl3):δ = 192, 167, 144, 134, 130,
129, 62, 46, 22, 14 ppm. (C12H14O3).
To a solution of 19.07 g (160 mmol, 4 Eq) dimethylformamide
dimethyl acetal (DMFA) in 150 mL of dry DMF was added 8.2 g
(40 mmol) of the β-keto ester 2 d). The solution was stirred at
reflux under N2 (g) for 48 h.The solvents were removed under
reduced pressure and by Kugelrohr distillation. The dark oily
product was purified by Kugelrohr distillation affording 8.88 g
(34.0 mmol) of a dark yellow oil, bp = 138–143 °C at 3 mm Hg,
in 85 % yield of E and Z isomers.TLC showed a single spot with
an Rf = 0.66 in 7:3 hexanes:ethyl acetate.IR (neat on NaCl):ν =
1742 cm–1 (C=O), 1678 cm–1 (C=O), 1634 cm–1 (C=O),
1607 cm–1 (aromatic C-C). 1H NMR of Z isomer (CDCl3): δ =
7.93, (s, 1 H), 7.58 (d, 2 H, J = 8.1 Hz), 7.10 (d, 2 H, J = 8.1 Hz),
3.90 (q, 2 H, J = 7.1 Hz), 2.86 (br.s., 6 H), 2.30 (s, 3 H), 0.87 (t,
3 H, J = 7.1 Hz) ppm. 13C NMR of Z isomer (CDCl3): δ = 194,
169, 155, 142, 139, 129.3, 129.0, 126, 60, 44, 22, 14 ppm.
(C15H19NO3).
Preparation of ethyl 1-(3Ј-chlorophenyl)-3-(dimethylamino)-
prop-2-enone-2-carboxylate (3 a)
To a solution of 21.2 g (0.178 mol, 4 Eq) dimethylformamide
dimethyl acetal (DMFA) in 175 mL of dry DMF was added
10.0 g (0.044 mol) of the β-keto ester (2 a). The solution was
stirred at reflux under N2 (g) for 18 h. The solvents were re-
moved under reduced pressure and by Kugelrohr distillation.
The dark oily product was purified by Kugelrohr distillation af-
fording 7.37 g (29.9 mmol) of a dark yellow oil, bp = 167 °C at
1.00 mm Hg, in 68 % yield.TLC showed a single spot with an Rf
= 0.66 in 7:3 hexanes:ethyl acetate.IR (neat on NaCl):ν = 1698
cm–1 (C=O), 1651 cm–1 (C=O).1H NMR of Z isomer (CDCl3):δ =
7.92 (s, 1 H), 7.30–7.82 (m, 4 H), 4.21 (q, 2 H, J = 7.2 Hz), 3.03
(br.s., 6 H), 1.25 (t, 3 H, J = 7.2 Hz) ppm. 13C NMR of Z isomer
(CDCl3): δ = 191.70, 167.48, 163.37, 157.09, 130.49, 129.72,
129.64, 129.61, 128.95, 127.76, 127.02, 62.04, 14.45 ppm.
(C14H16ClNO3).
Preparation of ethyl 3-(3Ј-chlorophenyl)-3-chloropropenal-
2-carboxylate (4 a)
To a solution of 5.47 g (22.2 mmol) of the vinylogous amide (3a)
in 100 mL CH2Cl2 was added 4.5 mL (7.4 g, 48 mmol, 2.2 Eq)
of phosphorus oxychloride.The solution was stirred at reflux un-
der N2 (g) for 3 h.The solvent was removed under reduced pres-
sure.To the resulting dark oil was added 100 mL of 1:1THF:H2O.
The solution was stirred at reflux overnight.The volatiles were re-
moved under reduced pressure and the aqueous layer was
washed 2× 50 mL with CHCl3.The combined CHCl3 layers were
washed 4× 50 mL with water.The CHCl3 layer was dried over an-
hydrous MgSO4, filtered, and the solvent removed under re-
duced pressure.The crude dark oil was purified by column chro-
matography on silica gel using 8:2 hexanes:ethyl acetate, Rf =
0.61. A pale yellow oil, 2.04 g (7.45 mmol), bp = 134 °C at
0.3 mm Hg, was afforded in 34 % yield as a mixture of E and Z
isomers. IR (neat on NaCl): ν = 1734 cm–1 (C=O), 1685 cm–1
(C=O), 1628 cm–1 (C=O), 1594 cm–1 (aromatic C-C). 1H NMR of
Z isomer (CDCl3):δ = 10.07 (s, 1H), 7.84 (s, 1H), 7.74 (d, 1H, J =
7.7 Hz), 7.25–7.41 (m, 2 H), 4.14 (q, 2 H, J = 7.2 Hz), 1.18 (t, 3 H,
J = 7.2 Hz) ppm. 13C NMR of Z isomer (CDCl3): δ = 187.62,
167.50, 137.90, 135.56, 134.06, 131.51, 130.50, 128.95,
128.39, 127.02, 62.04, 14.44 ppm. (C12H10Cl2O3).
Preparation of ethyl 1-(4Ј-chlorophenyl)-3-(dimethylamino)-
prop-2-enone-2-carboxylate (3 b)
To a solution of 19.10 g (0.160 mol, 4.1 Eq) dimethylform-
amide dimethyl acetal (DMFA) in 100 mL of dry DMF was add-
ed 8.77 g (0.039 mol) of the β-keto ester (2 b). The solution
was stirred at reflux under N2 (g) for 18 h.The solvents were re-
moved under reduced pressure and by Kugelrohr distillation.
The dark oily product was purified by Kugelrohr distillation af-
fording 10.10 g (0.0358 mol) of a dark yellow oil, bp = 181 °C at
0.77 mm Hg, in 93 % yield.TLC showed a single spot with an Rf
= 0.08 in 7:3 hexanes:ethyl acetate. IR (neat on NaCl): ν =
1727 cm–1 (C=O), 1683 cm–1 (C=O), 1631 cm–1 (C=O),
1587 cm–1 (aromatic C-C). 1H NMR of Z isomer (CDCl3): δ =
7.53–7.76 (m, 3 H), 7.29 (d, 2 H, J = 8.7 Hz), 3.90 (q, 2 H, J =
7.2 Hz), 2.92 (br.s., 6 H), 0.88 (t, 3 H, J = 7.2 Hz) ppm.13C NMR
of Z isomer (CDCl3): δ = 192.91, 168.79, 156.61, 130.61,
130.46, 129.92, 128.58, 128.17, 60.19, 14.34 ppm.
(C14H16ClNO3).
Preparation of ethyl 3-(4Ј-chlorophenyl)-3-chloropropenal-2-
carboxylate (4 b)
To a solution of 10.10 g (0.0358 mol) of the vinylogous amide
(3 b) in 100 mL CH2Cl2 was added 3.50 mL (5.76 g,
0.0375 mol, 1.05 Eq) of phosphorus oxychloride.The solution
was stirred at reflux under N2 (g) for 2 h. The solvent was re-
moved under reduced pressure. To the resulting dark oil was
added 100 mL of 1:1 THF:H2O. The solution was stirred at re-
flux for 18 h. The volatiles were removed under reduced pres-
sure and the aqueous layer was washed 3× 50 mL with ethyl
acetate. The combined ethyl acetate layers were washed 2×
50 mL with water.The ethyl acetate layer was dried over anhy-
drous MgSO4, filtered and the solvent removed under reduced
pressure. The crude dark oil was purified by column chroma-
tography on silica gel using 7:3 hexanes:ethyl acetate, Rf =
0.73.A pale yellow oil, 4.95 g (0.0181 mol), bp = 132–134 °C at
0.35 mm Hg, was afforded in 50.6 % yield of E and Z isomers.
IR (neat on NaCl): ν = 1732 cm–1 (C=O), 1682 cm–1 (C=O),
1590 cm–1 (aromatic C-C). 1H NMR of Z isomer (CDCl3): δ =
9.28 (s, 1 H), 7.42 (d, 2 H, J = 8.8 Hz), 7.32 (d, 2 H, J = 8.8 Hz),
4.31 (q, 2 H, J = 7.2 Hz), 1.29 (t, 3 H, J = 7.2 Hz) ppm.13C NMR
of Z isomer (CDCl3): δ = 186.07, 164.00, 153.88, 138.73,
136.34, 132.54, 131.54, 131.53, 129.61, 62.77, 14.45 ppm.
(C12H10Cl2O3).
Preparation of ethyl 1-(4Ј-bromophenyl)-3-(dimethylamino)-
prop-2-enone-2-carboxylate (3 c)
To a solution of 14.7 mL (13.2 g, 0.111 mol, 4.5 Eq) dimethyl-
formamide dimethyl acetal (DMFA) in 100 mL of dry DMF was
added 6.71 g (0.0247 mol) of the β-keto ester (2 c). The solu-
tion was stirred at reflux under N2 (g) for 18 h. The solvents
were removed under reduced pressure and by Kugelrohr distil-
lation. The dark oily product was purified by Kugelrohr distilla-
tion affording 5.35 g (16.4 mmol) of a dark yellow oil, bp =
181 °C at 1.10 mm Hg, in 66 % yield.TLC showed a single spot
with an Rf = 0.09 in 7:3 hexanes:ethyl acetate. IR (neat on
NaCl): ν = 1728 cm–1 (C=O), 1682 cm–1 (C=O), 1633 cm–1
(C=O), 1588 cm–1 (aromatic C-C). 1H NMR of Z isomer
(CDCl3): δ = 7.66 (s, 1 H), 7.56 (d, 2 H, J = 8.4 Hz), 7.45 (d, 2 H,
J = 8.4 Hz), 3.90 (q, 2 H, J = 7.2 Hz), 2.88 (br.s., 6 H), 0.87 (t,
3 H, J = 7.2 Hz) ppm.13C NMR of Z isomer (CDCl3):δ = 191.65,
167.42, 161.49, 155.31, 131.10, 130.68, 130.14, 129.79,
128.81, 58.80, 12.92 ppm. (C14H16BrNO3).

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 181–190
Ethyl 2-Phenacyl-3-phenylpyrrole-4-carboxylates 187
1700 cm–1 (C=O), 1591 cm–1 (aromatic C-C).1H NMR (CDCl3):
δ = 7.83 (d, 2 H, J = 8.3 Hz), 7.46 (d, 2 H, J = 8.3 Hz), 4.41 (s,
2 H) ppm. 13C NMR (CDCl3): δ = 192.10, 140.80, 133.06,
129.46, 129.43, 54.97 ppm. (C8H6ClN3O).
Preparation of ethyl 3-(4Ј-bromophenyl)-3-chloropropenal-2-
carboxylate (4 c)
To a solution of 6.44 g (19.7 mmol) of the vinylogous amide
(3 c) in 100 mL CH2Cl2 was added 1.84 mL (3.03 g,
19.7 mmol, 1 Eq) of phosphorus oxychloride.The solution was
stirred at reflux under N2 (g) for 4 h. The solvent was removed
under reduced pressure. To the resulting dark oil was added
100 mL of 1:1 THF:H2O. The solution was stirred at RT for
3 days, then at reflux for 5 h.The volatiles were removed under
reduced pressure and the aqueous layer was washed 3×
50 mL with ethyl acetate. The combined ethyl acetate layers
were washed 2× with 50 mL water.The ethyl acetate layer was
dried over anhydrous MgSO4, filtered, and the solvent removed
under reduced pressure.The crude dark oil was purified by col-
umn chromatography on silica gel using 8:2 hexanes:ethyl ace-
tate, Rf = 0.53. A pale yellow oil, 1.37 g (4.31 mmol), bp =
138–140 °C at 0.35 mm Hg, was afforded in 22 % yield of E
and Z isomers. IR (neat on NaCl): ν = 1736 cm–1 (C=O),
1676 cm–1 (C=O), 1584 cm–1 (aromatic C-C). 1H NMR of Z iso-
mer (CDCl3):δ = 9.28 (s, 1 H), 7.54 (d, 2 H, J = 8.7 Hz), 7.30 (d,
2 H, J = 8.7 Hz), 4.31 (q, 2 H, J = 7.2 Hz), 1.28 (t, 3 H, J =
7.2 Hz) ppm.13C NMR of Z isomer (CDCl3):δ = 187.21, 165.14,
155.09, 137.49, 134.18, 133.76, 132.83, 128.26, 63.92, 15.63
ppm. (C12H10BrClO3).
Preparation of 2-ammonium-4Ј-chloroacetophenone 4-tolu-
enesulfonate (7)
2-Azido-4Ј-choroacetophenone (6), 4.46 g (22.8 mmol),
triphenylphosphine, 5.98 g (22.8 mmol, 1 Eq), and p-tolue-
nesulfonic acid, 13.02 g, (68.46 mmol, 3 Eq) were added to
150 mL of THF and the mixture was stirred under N2 (g) at RT
overnight. The resulting white solid was filtered and washed
with THF affording 6.31 g (18.5 mmol) of pure salt, mp =
232–233 °C, in 81 % yield.TLC showed a single spot with an Rf
= 0.41 in 7:3 hexanes:ethyl acetate. IR (Nujol): ν = 3176, 3085,
3057 cm–1 (NH3), 1695 cm–1 (C=O), 1611 cm–1 (aromatic
C-C). 1H NMR (DMSO): δ = 8.20 (br. s., 3 H), 7.99 (d, 2 H, J =
8.7 Hz), 7.64 (d, 2 H, J = 8.7 Hz) 7.50 (d, 2 H, J = 8.1 Hz), 7.11
(d, 2 H, J = 8.1 Hz), 4.53 (s, 2 H), 2.28 (s, 3 H) ppm. 13C NMR
(CDCl3): δ = 192.22, 145.40, 139.90, 138.42, 132.70, 130.33,
129.49, 128.45, 125.85, 45.30, 21.02 ppm. (C15H16ClNO4S).
Preparation of ethyl 3-(3Ј-chlorophenyl)-2-phenacylpyrrole-4-
carboxylate (9 a)
1.48 (3.84 mmol) of the β-chloroenal (4 a) and 0.73 g
(4.23 mmol, 1.1 Eq) of α-aminoacetophenone hydrochloride
(8) were dissolved in 25 mL of dry DMF and stirred with heating
under N2 (g) for 3 days.The solvents were concentrated under
reduced pressure, then removed by Kugelrohr distillation. The
crude dark solid was purified by column chromatography on sil-
ica gel using 7:3 hexanes:ethyl acetate, Rf = 0.35, followed by
recrystallization in ethyl acetate/hexanes. Pale yellow crystals,
75 mg (0.21 mmol), mp = 135–137 °C, was afforded in 5.5 %
yield. IR (CDCl3 cell): ν = 3428 cm–1 (N-H), 2249 cm–1 (N-C),
1717 cm–1 (C=O), 1622 cm–1 (C=O), 1600 cm–1 (aromatic
C-C). 1H NMR (CDCl3): δ = 9.95 (br. s., 1H), 7.73 (d, 1H, J =
3.6 Hz), 7.32 (d, 2 H, J = 8.1 Hz), 7.23 (t, 2 H, J = 7.5 Hz),
6.99–7.11 (m, 4 H), 6.88–6.98 (m, 2 H), 4.15 (q, 2 H, J =
7.2 Hz), 1.16 (t, 3 H, J = 7.2 Hz) ppm. 13C NMR (CDCl3): δ =
187.98, 163.90, 137.60, 135.47, 133.27, 131.67, 130.28,
129.66, 129.01, 128.93, 128.86, 128.45, 127.97, 127.89,
127.42, 117.62, 60.32, 14.35 ppm. Elemental analysis
(C20H16ClNO3): C: 67.90 %, H: 4.56 %, 3.96 % (theoretical), C:
67.82 %, H: 4.46 %, N: 3.94 % (found).
Preparation of ethyl 3-chloro-3-(4Ј-methylphenyl)propenal-2-
carboxylate (4 d)
To a solution of 8.88 g (34.0 mmol) of the vinylogous amide
(3 d) in 100 mL CH2Cl2 was added 3.65 mL (6.00 g,
39.2 mmol, 1.2 Eq) of phosphorus oxychloride. The solution
was stirred at reflux under N2 (g) for 3 h. The solvent was re-
moved under reduced pressure. To the resulting dark oil was
added 100 mL of 1:1 THF:H2O. The solution was stirred at RT
for 3 days, then at reflux for 5 h.The volatiles were removed un-
der reduced pressure and the aqueous layer was washed 3×
with 50 mL ethyl acetate. The combined ethyl acetate layers
were washed 2× with 50 mL water.The ethyl acetate layer was
dried over anhydrous MgSO4, filtered, and the solvent removed
under reduced pressure.The crude dark oil was purified by col-
umn chromatography on silica gel using 8:2 hexanes:ethyl ace-
tate, Rf = 0.65. A pale yellow oil, 1.20 g (4.75 mmol), bp = 122–
123 °C at 0.3 mm Hg, was afforded in 14 % yield as a mixture
of E and Z isomers. IR (neat on NaCl): ν = 1735 cm–1 (C=O),
1602 cm–1 (aromatic C-C). 1H NMR of Z isomer (CDCl3): δ =
9.40 (s, 1 H), 7.43 (d, 2 H, J = 8.1 Hz), 7.29 (d, 2 H, J = 8.1 Hz),
4.41 (q, 2 H, J = 7.2 Hz), 2.44 (s, 3 H), 1.40 (t, 3 H, J = 7.2 Hz)
ppm.13C NMR of Z isomer (CDCl3):δ = 186.48, 164.24, 155.46,
142.94, 135.83, 131.55, 130.29, 129.80, 62.40, 21.70, 14.37
ppm. (C13H13ClO3).
Preparation of ethyl 3-(4Ј-chlorophenyl)-2-(4Ј-chlorophen-
acyl)pyrrole-4-carboxylate (9 b)
0.828 g (3.03 mmol) of the β-chloroenal (4b) and 1.03 g
(3.01 mmol, 1 Eq) of the α-aminoketone pTSA salt (7) was dis-
solved in 20 mL of dry DMF and stirred with heating under N2 (g)
for 3 days.The solvents were concentrated under reduced pres-
sure, then removed by Kugelrohr distillation.The crude dark solid
was purified by column chromatography on silica gel using 8:2
hexanes:ethyl acetate, Rf = 0.37, followed by recrystallization in
ethyl acetate/hexanes.Tan crystals, 46.5 mg (0.120 mmol), mp =
164–167 °C, was afforded in 4.0 % yield. IR (CDCl3 cell): ν =
3247 cm–1 (N-H), 2360 cm–1 (N-C), 1726 cm–1 (C=O),
1609 cm–1 (C=O), 1588 cm–1 (aromatic C-C).1H NMR (CDCl3):δ
= 9.69 (br. s., 1H, 7.73 (d, 1H, J = 3.4 Hz), 7.24 (d, 2 H, J =
6.7 Hz), 7.04 (d, 2 H, J = 6.1 Hz), 7.00 (d, 2 H, J = 6.7 Hz), 6.98
(d, 2 H, J = 6.1 Hz), 4.17 (q, 2 H, J = 7.2 Hz), 1.18 (t, 3 H, J =
7.2 Hz) ppm. 13C NMR (CDCl3): δ = 186.48, 163.74, 138.37,
135.83, 134.09, 132.73, 132.08, 131.88, 130.44, 130.03,
128.92, 128.21, 127.66, 117.64, 60.36, 14.43 ppm. Elemental
analysis (C20H15Cl2NO3): C: 61.87 %, H: 3.89 %, N: 3.61 % (theo-
retical), ): C: 61.94 %, H: 3.68 %, N: 3.47 % (found).
Preparation of 2-azido-4Ј-choroacetophenone (6)
To a stirring suspension of 2-bromo-4Ј-chloroacetophenone
(11.67 g, mmol) in 60 mL of DMSO at 10 °C was added 4.0 g
(61 mmol, 1.2 Eq) of sodium azide.The mixture was allowed to
come to RT and the resulting solution stirred at RT for 20 min.
The solution was added to 200 mL of H2O and the aqueous
mixture was washed 3× with 50 mL ethyl acetate. The com-
bined ethyl acetate layers were washed 3x with 50 mL water.
The organic layer was dried over anhydrous sodium sulfate, fil-
tered, and the solution was concentrated under reduced pres-
sure.The product was crystallized from the concentrated ethyl
acetate solution. The crystals were collected by suction filtra-
tion, washed with hexanes, and allowed to air dry. Yellow
needles, 7.06 g (36.1 mmol) were afforded in 72 % yield. mp =
68–69 °C. TLC showed a single spot with an Rf = 0.64 in 7:3
hexanes:ethyl acetate. IR (CDCl3 cell): ν = 2106 cm–1 (N3),

188 Evans et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 181–190
inhibition. Solid tumor cytotoxicity was determined utilizing
crystal violet/MeOH and read at 580 nm (Molecular
Devices) [10].
Preparation of ethyl 3-(4Ј-bromophenyl)-2-phenacylpyrrole-4-
carboxylate (9 c)
0.615 g (1.94 mmol) of the β-chloroenal (4c) and 0.367 g
(2.14 mmol, 1.1 Eq) of α-aminoacetophenone hydrochloride
(8) was dissolved in 25 mL of dry DMF and stirred with heating
under N2 (g) for 3 days.The solvents were concentrated under
reduced pressure, then removed by Kugelrohr distillation. The
crude dark solid was purified by column chromatography on
silica gel using 7:3 hexanes:ethyl acetate, Rf = 0.46, followed by
recrystallization in ethyl acetate/hexanes.Tan crystals, 132 mg
(0.332 mmol), mp = 152–154 °C, was afforded in 17 % yield.IR
(CDCl3 cell):ν = 3234 cm–1 (N-H), 2358 cm–1 (N-C), 1724 cm–1
(C=O), 1609 cm–1 (C=O).1H NMR (CDCl3):δ = 9.72 (br.s., 1 H),
7.69 (d, 1 H, J = 3.4 Hz), 7.24 (d, 2 H, J = 7.4 Hz), 7.21 (d, 2 H,
J = 7.4 Hz), 7.07 (m, 2 H, J = 8.5 Hz), 6.99 (t, 2 H, J = 7.4 Hz),
6.86 (d, 2 H, J = 8.5 Hz), 4.11 (q, 2 H, J = 7.2 Hz), 1.14 (t, 3 H,
J = 7.2 Hz) ppm.13C NMR (CDCl3):δ = 187.98, 163.97, 137.34,
133.07, 132.42, 132.15, 131.86, 130.48, 130.12, 129.13,
128.91, 128.00, 121.86, 117.23, 60.45, 14.56 ppm. Elemental
analysis (C20H16BrNO3):C:60.32 %, H:4.05 %, N 3.52 % (theo-
retical), C: 60.03 %, H: 3.82 %, N 3.55 % (found).
Incorporation studies
Incorporation of labeled precursors into 3H-DNA, 3H-RNA, and
3H-protein for 106 HL-60 leukemia cells was obtained [11] using
a concentration range of 25, 50, and 100 µM of the test agent
9 a over a 60 min incubation. The incorporation of 14C-glycine
(53.0 mCi/mmol) into purines [12] and the incorporation of
14C-formate (53.0 mCi/mmol) into pyrimidines [13] was deter-
mined in a similar manner.
Enzyme assays
Inhibition of various enzyme activities was performed by first
preparing the appropriate HL-60 leukemia cell homogenates or
subcellular fraction, then adding the drug to be tested during
the enzyme assay. For the concentration response studies, in-
hibition of enzyme activity was determined at 25, 50, and
100 µM of compound 9 a, after 60 min incubations. DNA
polymerase α activity was determined in cytoplasmic isolated
extracts [14] . Nuclear DNA polymerase β was determined by
isolating nuclei [15]. The polymerase activity for both α and β
was determined with 3H-TTP [16]. Messenger-, ribosomal-,
and transfer-RNA polymerase enzymes were isolated with dif-
ferent concentrations of ammonium sulfate; individual RNA
polymerase activities were determined using 3H-UTP [17, 18].
Ribonucleotide reductase activity was measured using 14C-
CDP with dithioerythritol [19]. The deoxyribonucleotides 14C-
dCDP were separated from the ribonucleotides by TLC on PEI
plates. Thymidine, TMP and TDP kinase activities were deter-
mined using 3H-thymidine (58.3 mCi/mmol) [20]. Carbamyl
phosphate synthetase activity was determined [21] with citrul-
line quantitated colorimetrically [22]. Aspartate transcarbamy-
lase activity was measured [21] and carbamyl aspartate was
quantitated colorimetrically [23].Thymidylate synthase activity
was analyzed by the 3H2O released which was proportional to
Preparation of ethyl 3-(4Ј-methylphenyl)-2-(4Ј-chlorophen-
acyl)pyrrole-4-carboxylate (9 d)
1.00 g (3.96 mmol) of the β-chloroenal (4 d) and 1.54 g
(3.96 mmol, 1 Eq) of the α-aminoketone pTSA salt (7) was dis-
solved in 20 mL of dry DMF and stirred with heating under N2
(g) for 66 h. The solvents were concentrated under reduced
pressure, then removed by Kugelrohr distillation. The crude
dark solid was purified by column chromatography on silica gel
using 7:3 hexanes:ethyl acetate, Rf = 0.22, followed by recry-
stallization in ethyl acetate/hexanes. White crystals, 221 mg
(0.601 mmol), mp = 155–157 °C, was afforded in 15 % yield.IR
(CDCl3 cell):ν = 3431 cm–1 (N-H), 2253 cm–1 (N-C), 1718 cm–1
(C=O), 1617 cm–1 (C=O).1H NMR (CDCl3):δ = 9.66 (br.s., 1 H),
7.64 (d, 1 H, J = 3.6 Hz), 7.14 (d, 2 H, J = 8.7 Hz), 6.88 (d, 2 H, J
= 8.7 Hz), 6.84 (d, 2 H, J = 8.0 Hz), 6.77 (d, 2 H, J = 8.0 Hz),
4.09 (q, 2 H, J = 7.2 Hz), 2.18 (s, 3 H), 1.11 (t, 3 H, J = 7.2 Hz)
ppm. 13C NMR (CDCl3): δ = 186.83, 163.94, 137.74, 137.57,
136.05, 133.88, 131.38, 130.45, 130.25, 130.02, 128.90,
128.13, 127.93, 117.60, 60.19, 21.30, 14.44 ppm. Elemental
analysis (C21H18ClNO3):C:68.57 %, H:4.93 %, N:3.81 % (theo-
retical), C: 68.25 %, H: 4.81 %, N: 3.77 % (found).
3
the amount of TMP formed from H-dUMP [24]. Dihydrofolate
reductase activity was determined by a spectrophotometric
method [25]. PRPP amidotransferase activity was determined
by the method of Spassova et al. [26]. IMP dehydrogenase ac-
tivity was analyzed with 8-14C-IMP (54 mCi/mmol) (Amersham,
Arlington Heights, IL) after separating XMP on PEI plates
(Fisher Scientific) byTLC [27]. Protein content was determined
for the enzymatic assays by the Lowry et al. technique [28].
Pharmacology and biochemical studies
Cytotoxicity
ct-DNA studies
Compounds 9 a–9 d (Table 1) were tested for cytotoxic activity
by homogenizing the drugs and preparing a 1 mg/mL solution
in 0.05 % Tween 80/H2O. These solutions were sterilized by
passing them through an acrodisc (0.45 µm).The following cell
lines were maintained by literature techniques [9]: murine L1210
lymphoid leukemia, and P388 lymphocytic leukemia, human
Tmolt3 and Tmolt4 acute lymphoblastic T cell leukemia, HL-60
leukemia, Hut-78 cutaneous lymphoma, THP-1 acute mono-
cytic leukemia, HCT-8 ileocecal adenocarcinoma, liver Hepe-2,
A-549 lung carcinoma, HSO osteosarcoma, KB epidermoid
nasopharynx, HeLa-S3 suspended cervical carcinoma, ovary
1-A9, SK-MEL-2 malignant melanoma, breast effusion MCF-7
and U-87-MG glioma.Normal human fibroblast 1788 were also
used to test cytotoxicity of the agents. The NCI protocol was
used to assess the cytotoxicity of the test compounds and
standards in each cell line. Values for cytotoxicity were ex-
pressed as ED50 = µg/mL, i.e. the concentration of the com-
pound inhibiting 50 % of cell growth. ED50 values were deter-
mined by the trypan blue exclusion technique [9]. A value of
less than 4 µg/mL was required for significant activity of growth
After deoxyribonucleoside triphosphates were extracted [29],
levels were determined by the method of Hunting and Hender-
son [30] with calf thymus DNA, E. coli DNA polymerase I, non-
limiting amounts of the three deoxyribonucleoside triphos-
phates not being assayed, and either 0.4 mCi of (3H-methyl)-
dTTP or (5-3H)-dCTP. The effects of compound 9 a on DNA
strand scission was determined by the methods of Suzuki et al.
[31], Pera et al. [32], and Woynarowski et al. [33]. HL-60 leuke-
mia cells were incubated with 10 µCi thymidine [methyl-3H,
84.0 Ci/mmol] for 24 h at 37 °C.HL-60 cells (107) were harvest-
ed and then centrifuged at 600 g × 10 min in PBS. They were
later washed and suspended in 1 mL of PBS. Lysis buffer
(0.5 mL; 0.5 M NaOH, 0.02 M EDTA, 0.01 % Triton X-100 and
2.5 % sucrose) was layered onto a 5–20 % alkaline-sucrose
gradient (5 mL; 0.3 M NaOH, 0.7 M KCl, and 0.01 M EDTA);
this was followed by 0.2 mL of the cell preparation. After the
gradient was incubated for 2.5 h at room temperature, it was
centrifuged at 12,000 RPM at 20 °C for 60 min (Beckman rotor
SW60). Fractions (0.2 mL) were collected from the bottom of

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 181–190
Ethyl 2-Phenacyl-3-phenylpyrrole-4-carboxylates 189
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the gradient, neutralized with 0.2 mL of 0.3 N HCl, and meas-
ured for radioactivity. Thermal calf thymus DNA denaturation
studies, ct-DNA UV absorption studies and DNA viscosity stud-
ies were conducted after incubation of compound 9 a at
100 µM at 37 °C for 24 h [34].
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Sample drugs were prepared in DMSO so that the stock final
concentration was 5 mM [w/v].The enzyme assay consisted of
test drugs at 50–200 µM, 1 unit of human DNA topoisomerase
II (p170 isoform) [TopoGen, Inc., Columbus, OH ], ca. 0.5 µg of
supercoiled PBR322 DNA in 50 mM Tris buffer, pH 7.5, 15 mM
β-mercaptoethanol, 30 mg/mL bovine serum albumin, 1 mM
ATP, 10 mM MgCl2, and 150 mM KCl [35].After 30 min incuba-
tion at 37 °C the reaction was terminated with 1 % SDS and
1 mg/mL proteinase K (v/v). After an additional hour of incuba-
tion, aliquots were applied to a 0.8 % agarose TBE gel (v/v)
containing 0.5 mg/mL ethidium bromide and 1 % SDS (w/v).
Following overnight electrophoresis at 30 V (constant), the gel
was destained and photographed using a UV-transilluminator
and Polaroid film. DNA topoisomerase I activity inhibition was
assayed by a similar method.The enzyme reaction consisted of
test drugs, 0.5 units of human topoisomerase I [TopoGen, Inc.,
Columbus, OH], 0.5 µg of supercoiled PBR322 DNA in 50 mM
Tris-HCl, pH 8.0, 100 mM KCl, 10 mM MgCl2, 2 mM 2-mer-
captoethanol, 30 µg/mL nuclease-free BSA.
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Flourogenic caspase assays
HL-60 cells (106) were incubated with test compound 9 a
(100 µM), VP-16 (50 µM), or 0.05 % tween 80 overnight. Cells
were collected at 600 × g for 3 min, washed with PBS (4 °C)
and re-suspended in 200 µL of lysis buffer (10 mM Hepes,
pH 7.4, 5 mM CHAPS, 5 mM DTT).Cells were allowed to swell
on ice for 10 min.then subjected to 3 cycles of freeze/thaw.Cel-
lular debris was removed by microfuging for 10 min. Aliquots
(10 µg) were added to 200 µL of reaction buffer (5 mM
HEPES, pH 7.4, 0.1 % CHAPS, 5 mM DTT, 2 µM caspase 3 or
caspase 8 substrate (Ac-DEVD-AMC or Ac-YVAD-AMC, Alexis
Biochemical).Additional reactions were performed in the same
manner but in the presence of inhibitor (Ac-DEVD-CHO or Ac-
YVAD-CHO, Alexis Biochemical.)
[14] H.Sawada, K.Tatsumi, M.Sadada, S.Shirakawa,T.Naka-
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Data is displayed in Tables and Figures as the means stand-
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control value. N is the number of samples per group. The Stu-
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International Union of Pure and
Applied Chemistry (IUPAC)
P. Heinrich Stahl / Camille G.Wermuth (Eds.)
Handbook of Pharmaceutical Salts
An essential step
in the preclinical phase
of drug development
Properties, Selection, and Use
2002. 388 pages. Hardcover. E 149.00* / sFr 220.00 / £ 85.00.
ISBN 3-906390-26-8.
*The e-Price is valid only for Germany.
O–
Cl
Na+
O
NH
Cl
Contents:
The majority of medicinal chemists in pharmaceutical industry whose primary focus is the
design and synthesis of novel compounds as future drug entities are organic chemists for whom
salt formation is often a marginal activity restricted to the short-term objective of obtaining
crystalline material. Because a comprehensive resource that addresses the preparation, selection,
and use of pharmaceutically active salts has not been available, researchers may forego the
opportunities for increased efficacy and improved drug delivery provided by selection of an
optimal salt.To fill this gap in the pharmaceutical bibliography, we have gathered an international
team of seventeen authors from academia and pharmaceutical industry who, in the contributions
to this volume, present the necessary theoretical foundations as well as a wealth of detailed
practical experience in the choice of pharmaceutically active salts.
The Physicochemical Background: Fundamentals of Ionic
Equilibria • Solubility and Dissolution of Weak Acids, Bases,
and Salts • Evaluation of Solid-State Properties of Salts •
Pharmaceutical Aspects of the Drug Salt Form • Biological
Effects of the Drug Salt Form • Salt-Selection Strategies • A
Procedure For Salt Selection and Optimization • Large-Scale
Aspects of Salt Formation: Processing of Intermediates and
Final Products • Patent Aspects of Drug-Salt Formation •
Regulatory Requirements for Drug Salts in the European
Union, Japan, and the United States • Selected Procedures
for the Preparation of Pharmaceutically Acceptable Salts of
Acids and Bases • Monographs on Acids and Bases
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