Cytotoxicity of substituted alkyl-3,4-bis(4-methoxyphenyl)pyrrole-2- carboxylates in L1210 lymphoid leukemia cells

Article abstract of DOI:10.1002/(SICI)1521-4184(199811)331:11<337::AID-ARDP337>3.0.CO;2-R

Two alkyl-3,4-bis(4-methoxyphenyl)pyrrole-2-carboxylates proved to be potent cytotoxic agents in the murine L1210 lymphoid leukemia screen. DNA synthesis was preferentially inhibited with the major target of the agents being de novo purine biosynthesis at the regulatory enzyme sites of PRPP- amido transferase and IMP dehydrogenase. Other enzymatic activities which were suppressed by the drugs were DNA polymerase α, RNA polymerases, ribonucleoside reductase and dihydrofolate reductase. The d[NTP] pools, nucleoside kinase and the pyrimidine pathway were not affected by the presence of drugs. The DNA molecule itself was not the target of the agents, i.e. no alkylation of nucleotide bases, intercalation between bases or cross- linking of DNA strands occurred. The agents did cause L1210 DNA fragmentation after 24 h incubation at 100 μM.

Full text of DOI:10.1002/(SICI)1521-4184(199811)331:11<337::AID-ARDP337>3.0.CO;2-R

Full Papers
Cytotoxicity of Substituted Alkyl-3,4-bis(4-methoxyphenyl)pyrrole-
2-carboxylates in L1210 Lymphoid Leukemia Cells
a)
a)
a)
a)
a)
a)
b)
Bruce S. Burnham , John T. Gupton , Keith Krumpe , T. Webb , Jordan Shuford , Brook Bowers , Amy E. Warren ,
b)
b)
Cheryl Barnes , and Iris H. Hall*
a) Department of Chemistry, University of North Carolina at Asheville, Asheville North Carolina, 28804, USA
b) Division of Medicinal Chemistry, School of Pharmacy, University of North Carolina, Chapel Hill , North Carolina 27599-7360, USA
Key Words: Alkyl-3,4-bis(4-methoxyphenyl)pyrrole-2-carboxylates; antineoplastic activity; purine synthesis inhibitors
[11]
acids , [(N-alkyl-1H,3H-1-oxoisoindolin-5-yl-oxyalkan-
Summary
[12]
oates , and 5,6-disubstituted 1(2)H-indazole- 4,7-di-
[13]
ones
. Recently, the marine natural product rigidin, which
Two alkyl-3,4-bis(4-methoxyphenyl)pyrrole-2-carboxylates
proved to be potent cytotoxic agents in the murineL1210 lymphoid
leukemia screen. DNA synthesis was preferentially inhibited with
the major target of the agents being de novo purine biosynthesis at
the regulatory enzyme sites of PRPP-amido transferase and IMP
dehydrogenase. Other enzymatic activities which were suppressed
by the drugs were DNA polymerase α, RNA polymerases, ribonu-
cleoside reductase and dihydrofolate reductase. The d[NTP] pools,
nucleoside kinase and the pyrimidine pathway were not affected
by the presence of drugs. The DNA molecule itself was not the
target of the agents, i.e. no alkylation of nucleotide bases, interca-
lation between bases or cross-linking of DNA strands occurred.
The agents did cause L1210 DNA fragmentation after 24 h incu-
bation at 100 µM.
is a pyrrolopyrimidine, has demonstrated several types of
biological activity
[14]
. Lamellarin O and Lukianol A also
represent new and related marine natural products which
possess a highly functionalized pyrrole nucleus. Further
modification has yielded 2,3- and 2,5-disubstituted pyrroles.
The purpose of this investigation is to explore two substituted
alkyl-3,4-bis(4-methoxyphenyl)pyrrole-2-carboxylates for
their antineoplastic activities since some similarity in struc-
tural features exist with the above compounds in that these
low molecular weight derivatives have been shown to be
potent antimetabolites in cancer cells.
Results
Introduction
Synthesis
Antineoplastic activity has been demonstrated by a number
[14]
Gupton and co-workers
in recent years have been ac-
of heterocyclic five membered ring structures. These include
[1]
tively involved in developing methods for the regiochemi-
cally controlled synthesis of polysubstituted pyrroles from
appropriately substituted derivatives of vinylogous iminium
salts. Pyrroles, compounds 1 and 2, were prepared as depicted
in Scheme 1 from the commercially available 2-(4-methoxy-
phenyl)-4′-methoxyacetophenone (3). This ketone is effi-
ciently converted to a vinylogous amide (4) by reaction with
N,N-dimethylformamide dimethyl acetal in N,N-dimethyl-
formamide. The vinyloglous amide (4) appeared to be a single
stereoisomer as determined by tlc and NMR and was used in
the next step without further purification. Subsequent treat-
ment of the crude vinylogous amide (4) with phosphorus
oxychloride in methylene chloride followed by hydrolysis of
the crude product in water/tetrahydrofuran produces the re-
spective β-chloroenal (5, a mixture of E and Z isomers with
the E isomer predominating) in good yield. The mixture
β-chloroenal is then reacted with either glycine methyl ester
or N-methylglycine ethyl ester in refluxing N,N-dimethyl-
formamide to produce the respective 2-carboalkoxy-3,4-
diarylpyrrole (1 or 2) in high overall yield. A complete
account of this new synthetic procedure is in preparation and
1-acyl- and 1,2-diacyl-4,4-diethyl-3,5-pyrazolidinediones
,
,
[2]
1-acyl- and 1,2-diacyl-1,2,4-triazolidinedione-3,5-diones
[3]
3,5-isoxazolidinediones and 2-isoxazoline-5-ones , 2-alk-
[4]
oxycarbonyl-5-aryl-1,3,5-triazine-4,6(1H,5H)diones , 3-
[5]
imino-1-oxaisoindolines , 5,6-substituted 1(2)H-indazole-
[6]
4,7-diones , N-pyridinyl and N-quinolinyl substituted
[7]
phthalimides and succinimides , diazomethyl ketone and
[8]
chloromethyl ketone analogues of N-tosyl amino acids ,
[9]
furan derivatives , and vinyl, cyanomethyl and pyranyl
[10]
activated esters of 2-furoic acid and 2-furylacrylic acids
,
[(N-alkyl-1,3-dioxo-1H,3H-isoindolin-5-yl)oxy]-alkanoic
3
will be published in the future. All compounds gave H NMR
and high resolution MS data consistent with their proposed
structures and were found to be greater than 95% pure as
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1998
0365-6233/98/1111/0337 $17.50 +.50/0

338
Hall and co-workers
36% to 39%, butdihydrofolate reductase activitywas reduced
more effectively by 78% and 53%. De novo purine synthesis
was reduced significantly at 31% and 41% with the activities
Table 2. Effects of compound 1 on L1210 lymphocytic leukemia cell
metabolism over 60 min.
——————————————————————————————
Percent of Control [X ± S.D]
Assay
Control 25µM
50µM
100µM
——————————————————————————————
DNA synthesis
RNA synthesis
Protein synthesis
DNA polymerase α
mRNA polymerase
rRNA polymerase
100±5a
100±6b
100±5c
100±6d
100±7e
100±4f
100±7g
100±5h
100±5i
100±5j
100±6k
100±5l
65±4*
110±5
74±4*
82±5
65±5*
82±5
43±4*
119±5
62±3*
53±4*
58±4*
76±4*
73±4*
66±3*
47±3*
76±4*
53±4*
39±4*
107±5
92±5
34±3*
124±6*
57±4*
38±3*
44±3*
59±4*
56±4*
61±4*
22±2*
79±5*
49±4*
35±3*
115±6
87±5
tRNA polymerase
84±6
Scheme 1. General synthetic route for the preparation of 2-carboalkoxy-3,4-
diarylpyrroles.
Ribonucleoside reductase
Dihydrofolate reductase
Purine de novo synthesis
PRPP amido transferase
IMP dehydrogenase
Pyrimidine de novo synthesis
69±4*
63±3*
87±4
61±4*
56±4*
determined by tlc analysis. It should also be pointed out that
compound 1 has previously been prepared by Furstner et
100±5m 104±4
[15]
al.
as part of the total synthesis of the marine natural
Carbamyl phosphate synthetase 100±7n
Aspartate transcarbamylase
Thymidylate synthetase
Thymine kinase
98±6
98±5
127±6* 149±6*
104±5
100±6
product Lukianol A; our material (1) had physical properties
100±6o
100±5p
100±6q
100±7r
99±6
108±7
92±5
113±7
72±4*
[15]
(mp, NMR) indentical to those previously reported
.
Compounds 1 and 2 afforded excellent cytotoxicity in the
murine L1210 lymphoid leukemic, P388 lymphocytic leuke-
107±6
78±4*
Thymidine monophosphate
kinase
mia, and human Tmolt and Tmolt T cell leukemia, HL-60
3
4
leukemia, HuT-8 lymphoma, THP-1 monocytic leukemia,
Thymidine diphosphate kinase 100±6s
92±6
117±6
147±5
105±5
99±6
59±4*
102±6
3
EH118 MG glioma and HeLa-S uterine carcinoma screens
d[ATP]
d[GTP]
d[CTP]
d[TTP]
100±5t
100±6u
100±5v
100±4w
with ED values less than 20 µM which compared well with
50
the clinical anti-neoplastic standards [Table 1].
The mode of action study in L1210 cells demonstrated that
DNA synthesis was suppressed by 64% and 43% at 100 µM
with less effect on RNA and protein syntheses after 60 min
incubation. Compound 1 caused a 24% elevation of RNA
syntheses with a 43% reduction of protein synthesis. Com-
pound 2 caused 28% reduction of RNA synthesis and 25%
reduction of protein synthesis. DNA polymerase α activity
was suppressed 62% and 56% , m-RNA polymerase was
reduced 56% and 42%, r-RNA polymerase was inhibited
41% and 31% and t-RNA polymerase activity was lowered
44% and 31% by compounds 1 and 2, respectively at 100 µM
after 60 min. Ribonucleoside reductase activity was reduced
——————————————————————————————
Control values for 106 cells over 60 min
* p ≤ 0.001
a 26152 dpm
b 4851 dpm
c 7461 dpm
d 47804 dpm
e 1502 dpm
f 4239 dpm
g 6400 dpm
h 2744 dpm
i 0.868 net OD units
j 92551 dpm
k 0.121 net OD units
l 76058 dpm
m 19758 dpm
n 0.392 mol citrulline
p 18463 dpm
q 1317 dpm
r 1179 dpm
s 1891 dpm
t 6.17 pmol
u 5.27 pmol
o 1.064 mol N-carbamyl v 6.87 pmol
aspartate w 6.94 pmol
Table 1. Cytotoxicity of Substituted Alkyl-3,4-bis(4-methyoxyphenyl)pyrrole-2-carboxylates.
—————————————————————————————————————————————————————————————–
ED50 values =µM
N=6
Mouse
L-1210 P388 Tmolt3 Tmolt4 HL-60 HuT-78 THP-1 HeLa-
Leukemia Leukemia Lymphoma Solid Colon Ileum Naso A549 M9812 Sarcoma Brain Skin
S3
Monocytic Uterine SW-480 HCT-8 pharynx Bone
Human
Adenocarcinoma KB
Lung
Osteo- Glioma Melanoma
Cp’d
#
—————————————————————————————————————————————————————————————–
1
8.87 4.29 5.12 4.47 5.00
7.78 3.30 3.63 6.50 4.61
3.81 2.96 10.97 22.71 15.61 14.61 19.49 16.92 15.85 27.89 7.39 21.88
2
4.59 2.89
8.38 20.40 10.62 14.23 17.37 16.52 18.27 25.75 10.30 22.15
8.76 19.49 14.06 10.44 11.68 25.82 25.33 3.54 7.73 26.89
25.77 106.76 96.37 23.27 69.29 116.88 94.40 37.73 29.84 120.3
Ara-C
9.99 9.797 10.98 2.36 3.90 11.76 2.54
35.10 17.09 58.77 6.68 5.22 50.88
Hydroxy-
urea
–
6MP
5FU
15.96 14.19 10.64 2.67 6.36 35.94 3.34 13.93 36.86 23.72 7.55 72.54 30.95 28.19 59.99 29.30 79.30
10.84 10.84 16.45 2.75 5.28 44.66 0.49 18.99 31.59 23.75 8.61 9.61 27.52 43.36 27.06 9.84 18.83
—————————————————————————————————————————————————————————————–
Standard deviation were all within 4% of the stated values. Ara-C = cytosine arabinoside; 6MP = 6-Mercaptopurine; 5FU = Fluorouracil
Arch. Pharm. Pharm. Med. Chem. 331, 337–341 (1998)

Alkyl-3,4-bis(4-methoxyphenyl)pyrrole-2-carboxylates
339
Table 3. Effects of compound 2 on L1210 leukemia cell metabolism over
60 min.
——————————————————————————————
TDP kinase activity by 47%. d[NTP] pool levels were gener-
ally not affected by the agents after 60 min except compound
1 caused a 41% reduction of d[CTP] pool levels [Tables 2
and 3].
Percent of Control [X ± SD]
(N=6) Assay
Control 25 µM 50 µM
100µM
Calf thymus-DNA studies demonstrated that the agents did
not directly interact with the DNA molecule based on DNA
viscosity, Tm values for DNA thermal denaturation and U.V.
absorption from 220 to 340 nm. L1210 DNA strand scission
studies showed that compound 1 and 2 did cause DNA strand
scission at 100 µM for 24 h [Fig 1].
——————————————————————————————
DNA synthesis
100±5
100±6 116±6
100±5 96±5
74±5*
69±4*
96±7
57±3*
72±4*
75±4*
44±3*
58±4*
69±4*
78±3*
64±5*
47±3*
49±4*
59±4*
37±3*
103±3
85±3
RNA synthesis
Protein synthesis
85±5
DNA polymerase α
mRNA polymerase
rRNA polymerase
100±6 101±5
100±7 136±6*
51±4*
69±4*
76±4*
79±4*
75±4*
74±4*
86±5
100±4
100±7
90±4
92±6
Discussion
tRNA polymerase
Ribonucleoside reductase
Dihydrofolate reductase
Purine de novo synthesis
PRPP amido transferase
IMP dehydrogenase
Pyrimidine de novo synthesis
100±5 108±6
Methyl 3,4 -bis(4-methoxyphenyl)pyrrole-2-carboxylate 1
and ethyl-N-methyl-3,4-bis(4-methoxyphenyl)pyrrole-2-
carboxylate 2 proved to effective cytotoxic agents in the a
number of leukemia and lymphoma screens. They were also
100±5
100±5
100±6
100±5
98±4
94±4
72±4*
91±6
61±3*
44±4*
106±5
87±4
3
active in the human HeLa-S uterine and glioma screens.
Compound 2 demonstrated selective activity against SW-480
colon adenocarcinoma growth but the compounds in general
were less active against the growth of solid tumors. Their
principal biochemical effect was inhibition ofL1210lympho-
cytic leukemia DNA synthesis with less effects on RNA and
protein synthesis. The inhibition of DNA synthesis appeared
to be the result of additive inhibitory effects of the agents on
several enzyme activities including DNA polymerase α, di-
hydrofolate reductase, PRPP-amido transferase and IMP de-
hydrogenase over 60 min. The suppression of the later three
enzyme activities would effectively reduce purine synthesis
in the cancer cells. The reduction in purines should reduce
DNA synthesis initially before RNA synthesis is affected
because deoxyribonucleotide pools are approximately 10%
of the ribonucleotide pools in mammalian cells. The marginal
reduction of ribonucleoside reductase activity by the agents
would further reduce deoxyribonucleotide pools for incorpo-
ration into DNA. The deoxyribonucleotide pools were not
actually reduced after 60 min incubation with the compounds.
This was probably a net effect since the agents inhibition
DNA polymerase α activity significantly. Not only would
this reduce DNA synthesis but the unincorporated d[NTP]s
would accumulate in the cell because they were not incorpo-
rated into the new strand of DNA. The agents appeared to
have minimum effect of pyrimidine de novo synthesis and on
nucleoside kinase activities. ct-DNA studies suggested that
the agents were not alkylating agents attacking the bases of
DNA, were not intercalators between base pairs of DNA and
did not cause DNA cross-linking. However, they did cause
DNA fragmentation which should be followed by reduction
in DNA synthesis and apoptosis. The biochemical effects and
concentration of these derivatives are typical of agents which
function as antimetabolites in cancer therapy.
100±5 109±6
Carbamyl phosphate synthetase 100±7
93±6
95±5
Aspartate transcarbamylase
Thymidylate synthetase
Tymidine kinase
100±6
92±6
91±4
100±5 100±6
121±7
83±6
151±6*
79±3*
73±4*
85±4
100±6
94±5
98±5
91±4
Thymidine monophosphate kinase 100±7
82±5
Thymidine diphosphate kinase
100±6
100±5
100±6
100±5
100±4
89±5
d(ATP)
d(GTP)
d(CTP)
d(TTP)
112±5
133±4
99±5
95±4
——————————————————————————————
of regulatory enzymes PRPP amido transferase being reduced
51% and 41% and IMP dehydrogenase being suppressed 65%
and 63% after 60 min at 100 µM. De novo pyrimidine syn-
thesis as well as the initial two regulatory enzymes, carbamyl
phosphate synthetase and aspartate transcarbamylase of the
pathway as well as thymidylate synthetase were not signifi-
cantly affected by the compounds after 60 min. Nucleoside
kinase activities were generally not reduced by the agents
except compounds 1 and 2 caused a 27% -28% reduction of
TMP kinase activity but compound 1 resulted in elevated
Acknowledgments
We gratefully acknowledge support of the synthetic work by an NIH
AREA grant (#R15CA67236-01A1) through the National Cancer Institute.
We wish to thank Professor Furstner for the copies of the NMR and MS
spectra of compound 1.
Figure 1. L1210 DNA strand scission after 24 h incubation at 100 µM of
compounds 1 and 2.
Arch. Pharm. Pharm. Med. Chem. 331, 337–341 (1998)

340
Hall and co-workers
126.70, 127.33, 128.49, 129.37, 131.13, 131.98, 158.01, 158.53, 162.11;
HR-MS (C22H23NO4, M + 1).
Experimental Part
Chemical Synthesis
Pharmacological Studies
Reagents: All chemicals were used as received from the manufacturer
(Aldrich Chemicals and Fisher). Nuclear magnetic resonance spectra were
obtained on a Varian Gemini 2000 NMR spectrometer in either CDCl3 or
d6-DMSO solution. Infrared spectra were recorded on a Perkin Elmer 1420
IR spectrometer as either nujol mulls or KBr pellets. High resolution mass
spectra were supplied by Dr. James A. Sikorski of Searle Corporation.
Melting points and boiling points were uncorrected.
Cytotoxicity
Compounds 1 and 2 (Table 1) were tested for cytotoxic activity by
homogenizing the drugs as a 1 mM 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 [17]: 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 monocytic leukemia, SW480 colorectal ade-
nocarcinoma, HCT-8 ileocecal adenocarcinoma, MB-9812 lung broncho-
genic, A-549 lung carcinoma, TE-418 osteosarcoma, KB epidermoid
nasopharynx, HeLa-S3 suspended and HeLa solid cervical carcinoma, Sk-
MEL-2 malignant melanoma and EH 118 MG glioma. The NCI protocol was
used to assess the cytotoxicity of the test compounds and standards in each
cell line. Values for cytotoxicity were expressed as ED50 = µM, i.e. the
concentration of the compound inhibiting 50% of cell growth. ED50 values
were determined by the trypan blue exclusion technique[17] . A value of less
than 20 µM was required for significant activity of growth inhibition. Solid
tumor cytotoxicity was determined utilizing crystal violet/MeOH and read
(E)-3-Dimethylamino-1,2-bis(4-methoxyphenyl)propenone (4)
To a solution of desoxyanisoin (5.00g, 19.5 mmol) in 100 ml DMF was
added 10.4 ml of N,N-dimethylformamide dimethyl acetal (9.30 g,
78.0 mmol). The solution was stirred under inert atmosphere at reflux for
18 h. The solvent was removed by Kugelrohr distillation affording a brown
solid (5.0g, 91%): mp = 114–117 °C ; tlc (1 spot) Rf = 0.56 (8:2 hexanes:ethyl
acetate); 1H NMR (CDCl3, 200 MHz) δ 2.73 (s, 6H), 3.78 (s, 6H), 6.73–6.82
(m, 4H), 7.05 (d, 2H, J = 9 Hz), 7.34 (s, 1H) , 7.42 (d, 2H, J = 9 Hz); HR-MS
(C19H21NO3, M+1) calcd 312.1600, found 312.1599.
(E)-3-Chloro-2,3-bis(4-methoxyphenyl)propenal (5)
at 580 nm (Molecular Devices)[18]
.
To
a solution of (E)-3-dimethylamino-1,2-bis(4-methoxyphenyl)-
propenone (0.11g, 0.35 mmol) in 10 ml of dichloromethane was added
3 drops of phosphorousoxychloride (~0.15g, 0.98 mmol). The solution was
stirred under inert atmosphere at reflux for 4 h. The solvent was removed
under reduced pressure. The crude residue was taken up in 20 ml of
THF:water [1:1] and stirred at room temperature for 24 h. The THF was
removed under reduced pressure. The aqueous mixture was washed 4 × 15 ml
with chloroform. The chloroform layers were combined and dried over
anhydrous magnesium sulfate, filtered and the solvent removed under re-
duced pressure affording a brown solid (0.100 g, 94%): mp 132–134 °C; tlc
(2 spots, E and Z isomers) Rf = 0.40 and 0.46 (8:2 hexanes: ethyl acetate); IR
(nujol) 1675 cm–1; 1H NMR (CDCl3, 200 MHz, major isomer, E ) δ 3.84 (s,
3H), 3.88 ( s, 3H), 6.97 (d, 4H, J = 9 Hz), 7.24 (d, 2H, J = 9 Hz),7.51 (d, 2H,
J = 9 Hz), 9.65 (s, 1H); HR-MS (C17H15ClO3, M+1) calcd 303.0788, found
303.0782.
Incorporation Studies
Incorporation of labeled precursors into 3H-DNA, 3H-RNA and 3H-protein
for 106 L1210 leukemia cells was obtained [19] using a concentration range
of 25 50 and 100 µM of the test agents over a 60 min incubations. The
incorporation of 14C-glycine (53.0 mCi/mmol) into purines[20] and the incor-
poration of 14C-formate (53.0 mCi/mmol) into pyrimidines[21] was deter-
mined in a similar manner.
Enzyme Assays
Inhibition of various enzyme activities was performed by first preparing
the appropriate L1210 cell homogenates or subcellular fractions, then adding
the drug to be tested during the enzyme assay. For the concentration response
studies, inhibition of enzyme activity was determined at 25, 50 and 100 µM
of compounds 1 and 2, after 60 min incubations. These concentrations of the
agents were selected for 1 hour incubation to establish the mode of action of
the derivatives quickly and are consistent with literature models [1–13] . DNA
Methyl 3,4 -bis(4-methoxyphenyl)pyrrole-2-carboxylate (1)
To a solution of (E)-3-chloro-2,3-bis(4-methoxyphenyl)propenal (79 mg,
0.26 mmol) in 10 ml dry DMF was added methyl glycinate hydrochloride
(54 mg, 0.43 mmol). The solution was stirred under inert atmosphere at reflux
for 20 h. The solvent was removed by Kugelrohr distillation. A crude black
residue was obtained which was purified by passing it through a short plug
of silica gel affording a light brown solid (72 mg, 82%) : mp 168–171 °C [lit
ref. 169–171 °C] ; tlc (1 spot) Rf = 0.24 (75:25 hexanes:ethyl acetate) . IR
(KBr) 3300 and 1670 cm–1; 1H NMR (CDCl3, 200 MHz) δ 3.73 (s, 3H), 3.76
(s, 3H), 3.82 (s, 3H), 6.75 (d, 2H, J = 9 Hz), 6.84 (d, 2 H, J = 9 Hz), 6.98–7.07
(m, 3H), 7.19 (d, 2H, J = 9 Hz), 9.18 (br s, 1H);13C NMR (CDCl3, 200 MHz)
δ 51.38, 55.23, 113.25, 113.80, 119.47, 120.41, 126.54, 126.63, 127.27,
129.24, 129.61, 132.04, 158.19, 158.71, 161.90; HR-MS (C20H19NO4,
M+1).
polymerase α activity was determined in cytoplasmic isolated extracts[22]
Nuclear DNA polymerase β was determined by isolating nuclei[23] . The
polymerase assay for both α and β was determined with 3H-TTP[24]
.
.
Messenger-, ribosomal- and transfer-RNA polymerase enzymes were iso-
lated with different concentrations of ammonium sulfate; individual RNA
polymerase activities were determined using 3H-UTP[25,26]. Ribonucleoside
reductase activitywasmeasuredusing14C-CDP with dithioerythritol[27]. The
deoxyribonucleotides 14C-dCDP were separated from the ribonucleotides by
TLC on PEI plates. Thymidine, TMP and TDP kinase activities were
determined using 3H-thymidine (58.3 mCi/mmol)[28]. Carbamyl phosphate
synthetase activity was determined [29] with citrulline quanititated colorimet-
rically[30] . Aspartate transcarbamylase activity was measured [29] and car-
bamyl aspartate was quanititated colorimetrically [31]. Thymidylate
3
synthetase activity was analyzed by the H2O released which was propor-
tional to the amount of TMP formed from 3H-dUMP [32]. Dihydrofolate
Ethyl N-Methyl-3,4-bis(4-methoxyphenyl)pyrrole-2-carboxylate (2)
[33]
reductase activity was determined by a spectrophotometric method
.
To a solution of (E)-3-chloro-2,3-bis(4-methoxyphenyl)propenal (a mix-
ture of isomers) (195 mg, 0.64 mmol) in 10 ml dry DMF was added ethyl
sarcosinate hydrochloride (139 mg, 0.91 mmol). The solution was stirred
under inert atmosphere at reflux for 20 h. The solvent was removed by
Kugelrohr distillation. A crude black residue was obtained which was
purified by passing it through a short plug of silica gel affording a yellow-
green oil (200 mg; 92%); bp 110–120 °C at 2.0 torr; tlc ( 1 spot) Rf = 0.56
PRPP amidotransferase activity was determined by the method of Spassova
et al.[34] . IMP dehydrogenase activity was analyzed with 8-14C-IMP
(54 mCi/mmol) (Amersham, Arlington Heights, IL) after separating XMP
on PEI plates (Fisher Scientific) by TLC[35] . Protein content was determined
for the enzymatic assays by the Lowry et al. technique [36]
.
(75:25 hexanes:ethyl acetate). IR (KBr) 1690 cm–1 1H NMR (CDCl3,
;
DNA studies
After deoxyribonucleoside triphosphates were extracted [37] , levels were
determined by the method of Hunting and Henderson [38] with calf thymus
DNA, E. coli DNA polymerase I, non-limiting amounts of the three deoxyri-
bonucleoside triphosphates not being assayed, and either 0.4 mCi of (3H-
200 MHz) δ 1.02 (t, 3H, J = 7 Hz), 3.74 (s, 3H), 3.81 (s, 3H), 3.96 (s, 3H),
4.08 (q, 2H, J = 7 Hz), 6.73 (d, 2H, J = 9 Hz), 6. 83 (d, 2H, J = 9 Hz), 6.88
(s, 1H), 7.00 (d, 2H, J = 9 Hz), 7.13 (d, 2H, J = 9 Hz), 13C NMR (CDCl3,
200 MHz) δ 13.93, 37.66, 55.23, 59.75, 113.06, 113.74, 120.72, 123.96,
Arch. Pharm. Pharm. Med. Chem. 331, 337–341 (1998)

Alkyl-3,4-bis(4-methoxyphenyl)pyrrole-2-carboxylates
341
methyl)-dTTP or (5-3H)-dCTP. The effects of compounds 1 and 2 on DNA
strand scission was determined by the methods of Suzuki et al. [39] , Pera et
[16] W.Y. Yoshida, K.K. Lee, A.R. Carroll, P.J. Scheuer, Helv. Chim. Acta
1992, 75, 1721–1725.
[40]
[41]
al.
and Woynarowski et al.
. L1210 leukemia cells were incubated
[17] R.J. Geran, N.M. Greenberg, M.M. Macdonald, A.M. Schumacher, B.J.
with 10 Ci thymidine [methyl-3H, 84.0 Ci/mmol] for 24 h at 37°C. L1210
cells (107) were harvested 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 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 the gradient, neutralized with 0.2 ml of
0.3 N HCl, and measured for radio-activity. Thermal calf thymus DNA
denaturation studies, DNA u.v. absorption studies and DNA viscosity studies
were conducted after incubation of compounds 1, and 2 at 100 µM at 37°C
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Received: December 19, 1997 [FP269]
Arch. Pharm. Pharm. Med. Chem. 331, 337–341 (1998)