Pharmacophore Hybridization to Discover Novel Topoisomerase II Poisons with Promising Antiproliferative Activity

Article abstract of DOI:10.1021/acs.jmedchem.7b01388

We used a pharmacophore hybridization strategy to combine key structural elements of merbarone and etoposide and generated new type II topoisomerase (topoII) poisons. This first set of hybrid topoII poisons shows promising antiproliferative activity on hu

Full text of DOI:10.1021/acs.jmedchem.7b01388

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Brief Article
Pharmacophore hybridization to discover novel Topoisomerase
II poisons with promising antiproliferative activity
José-Antonio Ortega, Laura Riccardi, Elirosa Minniti, Marco Borgogno, Jose M.
Arencibia, Maria Laura Greco, Anna Minarini, Claudia Sissi, and Marco De Vivo
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01388 • Publication Date (Web): 27 Oct 2017
Downloaded from http://pubs.acs.org on October 29, 2017
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Pharmacophore hybridization to discover novel Topoisomerase II
poisons with promising antiproliferative activity
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Jose Antonio Ortega,a,# Laura Riccardi,a,# Elirosa Minniti,a,c Marco Borgogno,a Jose M. Arencibia,a Ma-
ria L. Greco,b Anna Minarini,c Claudia Sissi,b,* and Marco De Vivoa,d,*
a Laboratory of Molecular Modeling and Drug Discovery. Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova (Ita-
ly). E-mail: marco.devivo@iit.it
b Department of Pharmaceutical and Pharmacological Sciences. University of Padova, Via Marzolo 5, 35131 Padova (Italy)
E-mail: claudia.sissi@unipd.it
c Department of Pharmacy and Biotechnology. Alma Mater Studiorum-University of Bologna, Via Belmeloro 6, 40126 Bo-
logna (Italy)
d IAS-5 / INM-9 Computational Biomedicine Forschungszentrum Jülich Wilhelm-Johnen-Straße 52428 Jülich (Germany)
# Equally contributed.
KEYWORDS topo II poisons, merbarone, etoposide, anticancer drug.
ABSTRACT: We used a pharmacophore hybridization strategy to combine key structural elements of merbarone and etoposide,
and generate new type II topoisomerase (topoII) poisons. This first set of hybrid topoII poisons shows promising antiproliferative
activity on human cancer cells, endorsing their further exploration for anticancer drug discovery.
INTRODUCTION
introduced key pharmacophoric elements of etoposide (E
ring)11 and merbarone (thiobarbituric core)12 into a new hybrid
scaffold (Figure 1). Importantly, compared to the template
compounds, our new compounds show boosted potency in
blocking topoII function as well as promising antiproliferative
activity against human cancer cells.
Human type II topoisomerase (topoII) enzymes are essential
for DNA topology modification, and are a validated anticancer
drug target.1–3 Several topoII inhibitors are clinically available,
but drug resistance and the severe side-effects of topoII-
targeted drugs are an issue. Novel and safer topoII inhibitors
for better anticancer therapeutics are thus required.4–6
In detail, these new hybrid molecules were generated based
on merbarone’s thiobarbituric core, which was modified as
follows: 1) introduction of either two ethyl groups or phenyl
rings at each nitrogen of the thiobarbituric core. Lipophilic
substituents at those nitrogen atoms have been shown to im-
prove merbarone’s cell permeability;18–20 2) functionalization
of the phenyl ring in merbarone to mimic the pendant aromatic
ring (E-ring) of etoposide, which was demonstrated to be es-
sential to block topoII activity.11 This modification was
planned to fine-tune the drug-target interactions at the
topoII/DNA cleavage complex, as suggested by molecular
TopoII inhibitors are divided into different classes accord-
ing to either their chemical scaffold or their molecular mecha-
nism of action.7–10 ‘TopoII poisons’ is the term for topoII tar-
geted agents that act by trapping the covalent topoII/DNA
cleavage complex, which is formed during the catalytic cycle
required for DNA topology modulation. One notable topoII
poison is the chemotherapy drug, etoposide (Figure 1). It was
the first successful anticancer agent to target topoII, and is still
used to treat a variety of cancer types, despite the chances of
severe side-effects.4,11
Merbarone is another well-known topoII blocker and one of
the first and most promising topoII inhibitors (Figure 1).12
Merbarone is a thiobarbituric derivative (6-hydroxy-4-oxo-N-
phenyl-2-thioxo-1H-pyrimidine-5-carboxamide) that impairs
cell cycle and proliferation of different cancer cell lines.12
Merbarone underwent clinical trials as a treatment for various
types of cancer.13,14 However, these trials were halted due to
insufficient anticancer activity and nephrotoxicity issues.15
That is, this promising anticancer chemical scaffold did not
generate the expected efficacy in vivo.
Figure 1. TopoII poisons designed via a pharmacophore hybrid-
ization strategy. The new hybrid functional scaffold (center) is
obtained by merging merbarone’s thiobarbituric core, with dif-
ferent decorations of the two heterocycle nitrogen atoms (left),
with the E-ring of etoposide (right).
RESULTS AND DISCUSSION
Here, we report on the design, synthesis, and initial biologi-
cal evaluation of a first set of new compounds designed via a
pharmacophore hybridization strategy.16,17 Specifically, we
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The molecular mechanisms leading to the topoII inhibition
Scheme 1. 2- Thiobarbituric acid derivatives synthesis.
1
by the five most active compounds, 1-5, were further charac-
terized (see Table 1). UV measurements showed that DNA
alone does not produce variations of the ligand absorbance in-
duced by DNA. This suggests a lack of relevant binding of all
our new derivatives to the DNA alone. Consistently, as al-
ready observed with merbarone,12, 26 CD titration of ctDNA
with the compounds presented in this work showed their ina-
bility to alter DNA structural arrangement in the absence of
topoII. As a consequence, this excludes an interference in the
DNA-topoII ligation step as a mechanism of inhibition. Intri-
guingly, these N,NI -diphenyl derivatives seemed to act differ-
ently than merbarone, while behaving similarly to etoposide.
In fact, while merbarone is not a poison,12, 26 some compounds
in the present work containing merbarone’s thiobarbituric core
showed the ability to stabilize the topoII/DNA cleavage com-
plex, thus acting as a poison. This was demonstrated by cleav-
age assay with compounds 1-3 and 5, which showed the pres-
ence of linear DNA, although to a lesser extent than etoposide
(see Table 1 and Figure S5 at SI) as a consequence of cleava-
ble complex stabilization. Notably, this evidence sustains our
docking calculations at the etoposide binding site.11, 23 In con-
trast, 4’s poor potency in blocking topoII was reflected in its
inability to generate the topoII/DNA cleavage complex.
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docking (see data below and in SI). The resulting hybrid scaf-
fold was synthesized using a general procedure for amide de-
rivatives synthesis (Scheme 1).21 We thus obtained 16 new
hybrid compounds with different functionalizations (see SI for
complete list).
Initially, all compounds were tested against human topoII in
a relaxation inhibition assay (see SI). The N,Ni-diphenyl deriv-
atives (compounds 1-8) were consistently much more active
than the corresponding diethyl ones (9-16, see SI). Table 1 re-
ports the IC50 values for the most active N,NI-diphenyl deriva-
tives (1-5, Scheme 1, see SI for compounds 6-16). The im-
proved activity of our N,NI -diphenyl (vs N,NI -diethyl) deriva-
tives can be rationalized by their predicted binding mode at the
cleavage site. Indeed, molecular modelling and docking22, 23
showed that N,NI -diphenyl derivatives formed good contacts
with topoII residues located in front of both DNA grooves
(Asp479 and Arg503 - more on this below). Thus, compared
to the corresponding diethyl derivatives, N,NI -diphenyl deriv-
atives likely act as a better linker between distal topoII/DNA
structural elements, mimicking etoposide’s binding mode (see
SI). This is further corroborated by the experimentally meas-
ured cleavage product formation, which was only generated by
N,NI -diphenyl derivatives (see Table 1 and SI)
Thus, our experimental findings indicate that compounds 1-
3 and 5 act by poisoning the topoII/DNA cleavage complex,
similarly to etoposide. In addition, our docking calculations
further support the hypothesis that our active hybrid topoII
poisons may favor the formation of a stable topoII/DNA
cleavage complex. Indeed, our docking results suggest key
interactions between the N,NI -diphenyl derivatives and the
topoII/DNA complex. In particular, 1 is predicted to have a
binding mode very similar to that of etoposide (Figure 2), with
its 3,5-dimethoxy-4-phenol ring forming an H-bond with
Asp479. Also, one of the two unsubstituted phenyl rings of 1
was extended toward the binding site that is occupied by the
glycosidic moiety of etoposide (Figure 2).11 The other phenyl
ring overlapped with the D-ring of etoposide, thus closer to the
scissile phosphate of the T+1 nucleotide along the cleaved
DNA strand (Figure 2). Interestingly, this binding mode of the
two naked phenyl rings was conserved in 4 and 5, too. How-
ever, the 3-phenol ring of 4 interacted with the side chain of
Arg503, pointing its hydroxyl group toward the DNA back-
bone. For this reason, the binding pose of 4 did not allow an
extension of the 3-phenol ring into the DNA minor groove, as
shown for compounds 1-3 and 5. This may explain 4’s inabil-
ity to stabilize the topoII/DNA cleavage complex.
Compound 1, which bears a 3,5-dimethoxy-4-phenol frag-
ment (like the E-ring in etoposide), was equipotent to mer-
barone (Table 1), with an IC50 of 120 µM. Replacing the hy-
droxyl group of 1 with a methoxy lead to a complete loss of
activity (compound 6, see SI). An equally negative effect was
observed when the same hydroxyl group in 1 was removed
(compound 7, see SI). Compound 2, in which the two methoxy
groups were removed, was slightly worse than 1, with an IC50
of 150. These results are in agreement with previous studies
that demonstrate the crucial role of the etoposide E-ring sub-
stituents for topoII inhibition.24,25
Interestingly, conserving only one methoxy group in the
meta- position, as in 3, returned an IC50 of 30 µM, with a 4-
fold increase of potency compared to both merbarone and
etoposide. Replacing this methoxy in 3 with a hydroxyl group,
as in 4, significantly decreased topoII inhibitory activity (IC50
200 µM). Finally, a naked phenyl ring, as in 5, showed an IC50
of 5 µM, which is 24-fold better than merbarone and etopo-
side, although the topoII/DNA cleavage complex formation
was reduced (see below).
Figure 2. Docking of compounds 1 and 3 (yellow) in the topoII
(gray) /DNA (cyan) cleavage complex, and their superposition
11
11
I
to tthheeccryrystsatal lpopsoeseofoeftoeptospiodseide(a(lPlDNB,N-3dQipXh3e)nyl (daellrivNa,tNives-
idnipShIe)n. yl derivatives in SI).
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Table 1. Data on compound potency and properties.
1
2
3
4
5
OMe
OH
OH
OH
N
O
O
OH
N
O
O
OH
N
O
O
OH
N
O
O
OH
N
O
O
N
N
H
OMe
N
N
H
N
N
H
OMe
N
N
H
OH
N
N
H
S
S
S
S
S
6
7
8
9
14
25
36
HeLae
47
A549e
53
DU145e
MCF7e
(µM)
TopoII
inhibitiona (µM) complexb (µM)
Cleavage
DNA
Interactionc
Minor
grooved
Compound
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(µM)
(µM)
(µM)
Etoposide
120 ±10
120 ± 12
120 ± 15
150 ± 19
30 ± 6
20
no
no
no
no
no
no
no
yes
no
2.4±0.9
10.5±4.0
83.9±3.0
53.5±1.9
66.1±7.5
14.0±0.9
94.2±0.7
13.6±0.8
1.3±0.1
40.0±2.7
17.5±1.1
37.1±3.7
6.9±0.2
59.4±6.1
9.2±0.3
1.0±0.4
Merbarone
no
62.3±6.4
18.1±0.8
42.9±6.1
8.5±0.5
18.9±2.0
26.7±0.1
42.5±7.7
12.5±1.5
49.6±2.7
16.8±0.8
1
2
3
4
5
200
200
100
no
yes
yes
yes
no
200 ± 22
39.1±1.3
5 ± 1.0
b
200
yes
10.8±0.2
aIC50 values. Compound concentration necessary to observe the same amount of topoII/DNA cleavage complex generated by
etoposide at 20 µM. cInteraction with DNA in absence of topoII enzyme. dExtension of the functionalized group of the hybrid com-
pound to the DNA minor groove, compared to the E-ring of etoposide. eAntiproliferative activity for each cellular line.
Moreover, the two naked phenyl rings of 3 protruded into
the DNA major groove, in front of the T+1/A+4 and C-1/G+5
base pairs, interacting with the side chains of Met782 and
Met778. In this way, the methoxy group of 3 was between the
side chain of Arg503 and the deoxyribose of the T+1 nucleotide
(Figure 2). Thus, the methoxy group forms key topoII/DNA
interactions in 3, likely explaining its slightly higher ability to
form topoII/DNA cleavage complex (100 for 3 vs 200 µM for
all the other new compounds). Notably, the relevance of this
methoxy group is well-known for etoposide, where its removal
leads to a significant loss of activity.11,24,25
EXPERIMENTAL SECTION
General considerations. All the commercial available rea-
gents and solvents were used as purchased from vendors with-
out further purification. Dry solvents were purchased from
Sigma-Aldrich. Automated column chromatography purifica-
tions were done using a Teledyne ISCO apparatus (Com-
biFlash® Rf) with pre-packed silica gel columns of different
sizes (from 4 g up to 24 g) and mixtures of increasing polarity
of cyclohexane and ethyl acetate (EtOAc) or dichloromethane
(DCM) and methanol (MeOH). NMR experiments were run
on a Bruker Avance III 400 system (400.13 MHz for 1H, and
100.62 MHz for 13C), equipped with a BBI probe and Z-
gradients. Spectra were acquired at 300 K, using deuterated
dimethylsulfoxide (DMSO–d6) or deuterated chloroform
Finally, we evaluated the antiproliferative activity of these
compounds in human endometrial (HeLa), breast (MCF7),
lung (A549), and androgen-independent prostate (DU145)
cancer cell lines. Overall, we found a general improvement of
the antiproliferative activity of these new compounds com-
pared to merbarone, with an increase in potency, up to 6-fold
in HeLa, MCF7, and A549 cell lines. These compounds were
also slightly more potent than merbarone in DU145 cells (Ta-
ble 1). Compared to etoposide, they were equipotent (MCF7)
or slightly less active (HeLa, A549 and DU145). Notably, we
also observed an increase in H2AX phosphorylation after
treatment of HeLa cells with compound 5 (Figure S7),27 which
indicates that the cytotoxicity observed is likely caused by cel-
lular inhibition of topoII.
1
(CDCl3) as solvents. For H-NMR, data are reported as fol-
lows: chemical shift, multiplicity (s= singlet, d= doublet, dd=
double of doublets, t= triplet, q= quartet, m= multiplet), cou-
pling constants (Hz) and integration. UPLC/MS analyses were
run on a Waters ACQUITY UPLC/MS system consisting of a
SQD (single quadrupole detector) mass spectrometer equipped
with an electrospray ionization interface and a photodiode ar-
ray detector. The PDA range was 210–400 nm. Analyses were
performed on an ACQUITY UPLC BEH C18 column
(100x2.1mmID, particle size 1.7 µm) with a VanGuard BEH
C18 pre-column (5x2.1 mmID, particle size 1.7 µm). Mobile
phase was 10 mM NH4OAc in H2O at pH 5 adjusted with
CH3COOH (A) and 10 mM NH4OAc in CH3CN–H2O (95:5)
at pH 5.0. The mobile-phase B proportion increased from 10
% to 90 % in 6.5 min with a 0.5 ml/min flow rate. Elec-
trospray ionization in positive and negative mode was applied.
ESI was applied in positive and negative mode. All tested
compounds showed ≥ 95 % purity by NMR and UPLC/MS
analysis.
CONCLUSION
In summary, we present new human topoII poisons obtained
by merging, into a single new hybrid functional scaffold, key
pharmacophoric elements of etoposide and merbarone. Im-
portantly, we obtained new compounds that are significantly
active in blocking topoII function, including when compared
to the two template structures. These new hybrid molecules
show promising antiproliferative activity against human can-
cer cells. Thus, taken together, these results endorse a further
exploration of this first set of new hybrid compounds to better
characterize their mechanism of action and their overall poten-
tial as novel anticancer therapeutics.
4-hydroxy-N-(4-hydroxy-3,5-dimethoxy-phenyl)-6-oxo-1,3-
diphenyl-2-thioxo-pyrimidine-5-carboxamide (1).
Titled compound was synthesized following the general
procedure B (see SI) using compound II (200 mg, 0.51 mmol)
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1
and 4-amino-2,6-dimethoxy-phenol (63.8 mg, 0.51 mmol) ob-
taining 15.7 mg of pure compound (yield 16 %). Synthesis of
the intermediates compound II and 4-amino-2,6-dimethoxy-
phenol are in the SI. Rt = 1.96 min; MS (SI) m/z: 492.1 [M-
, [M-H]- calculated: 414.1. H NMR (400 MHz, DMSO-d6) δ
11.65 (s, 1H), 7.57 – 7.51 (m, 2H), 7.50 – 7.46 (m, J = 7.6 Hz,
3H), 7.43 – 7.37 (m, J = 7.3 Hz, 4H), 7.36 – 7.31 (m, J = 7.8
Hz, 4H), 7.24 (t, J = 7.4 Hz, 1H). 13C NMR (101 MHz,
CDCl3) δ 178.67 (CONH), 169.32 (CS), 167.82 (COH),
162.32 (CO), 139.22 (C), 138.14 (C), 135.21 (C), 129.88
(CH), 129.79 (CH), 129.51(CH), 129.39(CH), 129.15(CH),
128.75(CH), 128.63(CH), 126.58(CH), 122.08(CH), 83.61(C).
qNMR: 95.3 %.
1
2
3
4
5
6
7
8
1
H]+, [M-H]- calculated: 492.1. H NMR (400 MHz, CDCl3):
11.82 (s, 1H), 7.58 – 7.47 (m, 6H), 7.31 (dd, J = 7.4, 3.1 Hz,
4H), 6.76 (s, 2H), 5.47 (s, 1H), 3.86 (s, 6H). 13C NMR (101
MHz, CDCl3) δ 178.59 (CONH), 168.63 (CS), 162.39 (C),
147.22 (C), 139.26 (C), 138.16 (C), 137.04 (C), 133.42 (C),
129.87 (CH), 129.76 (CH), 129.36 (CH), 129.15 (CH), 128.77
(CH), 128.63 (CH), 127.34 (CH), 99.60 (CH), 56.59 (OCH3).
qNMR: 95.3 %.
9
ASSOCIATED CONTENT
Supporting Information.
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This material is available free of charge via the Internet at
http://pubs.acs.org.
Molecular formula strings and additional experimental data (CSV)
Summary reporting all the synthesized compounds (Table S1)
Detailed synthetic procedures for compounds 1 to 16 and corre-
sponding intermediates.
4-hydroxy-N-(4-hydroxyphenyl)-6-oxo-1,3-diphenyl-2-
thioxo-pyrimidine-5-carboxamide (2).
Titled compound was synthesized following the general
procedure B (see SI) using compound II (100 mg, 0.27 mmol)
and 4-aminophenol (30.0 mg, 0.27 mmol) obtaining 25.1 mg
of pure compound (yield 22 %). Rt = 2.10 min; MS (SI) m/z:
H-NMR, C-NMR, mass spectrometry and chromatographic char-
acterization of compounds 1 to 16.
1
432.1 [M-H]-, [M-H]- calculated: 432,1. H NMR (400 MHz,
DMSO-d6) δ 11.49 (s, 1H), 9.64 (s, 1H), 7.48 (t, J = 7.5 Hz,
4H), 7.40 (t, J = 7.3 Hz, 2H), 7.34-7.32 (m, J = 8.2, 3.2 Hz,
6H), 6.78 (d, J = 8.8 Hz, 2H). 13C NMR (101 MHz, DMSO-d6)
δ 178.40 (CS), 168.12 (CONH), 155.76 (C), 139.19 (C),
129.16 (CH), 128.98 (CH), 128.39 (CH), 124.02 (CH), 115.69
(CH), 83.59 (C). qNMR: 95.0 %.
Summary of compounds 1 to 16 activity results (Table S2)
The structural model of topoII (Figure S1, S2)
Docking pose of compounds 1-5 (Figure S3)
Topoisomerase inhibition, Topoisomerase poisoning and ctDNA
binding assays procedures.
Cell lines and cell viability assay description.
4-hydroxy-N-(3-methoxyphenyl)-6-oxo-1,3-diphenyl-2-
thioxo-pyrimidine-5-carboxamide (3).
AUTHOR INFORMATION
Corresponding Authors
Titled compound was synthesized following the general
procedure B (see SI) using compound II (100 mg, 0.27 mmol)
and m-anisidine (0.031 ml, 0.27 mmol) obtaining 66.1 mg of
pure compound (yield 55 %). Rt = 2.10 min; MS (SI) m/z:
*Phone: +39 010 71781 577 E-mail: marco.devivo@iit.it
*Phone: +39 049 827571 E-mail: claudia.sissi@unipd.it
1
446.1 [M-H]+, [M-H]+ calculated: 446,1. H NMR (400 MHz,
Author Contributions
CDCl3) δ 11.85 (s, 1H), 7.62 – 7.52 (m, 4H), 7.52 – 7.45 (m,
2H), 7.40 – 7.28 (m, 4H), 7.27 (d, J = 4.4 Hz, 2H), 7.13 – 7.01
(m, 2H), 6.78 (dd, J = 8.2, 2.4 Hz, 1H), 3.79 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ 169.33 (CONH), 167.83 (C), 160.36 (C),
139.22 (C), 136.31 (C), 130.26 (CH), 129.87 (CH), 129.77
(CH), 129.38 (CH), 129.14 (CH), 128.75 (CH), 128.63 (CH),
114.25 (CH), 112.31 (CH), 107.77 (CH), 83.65 (C), 55.57
(OCH3). qNMR: 96.1 %.
The manuscript was written through contributions of all authors. /
All authors have given approval to the final version of the manu-
script. #These authors contributed equally.
Notes
The authors declare the following competing financial interest(s):
One patent application protecting the class of compounds dis-
closed in this manuscript has been filed by the following authors:
Marco De Vivo, Jose Antonio Ortega, Jose M. Arencibia and
Claudia Sissi.
4-hydroxy-N-(3-hydroxyphenyl)-6-oxo-1,3-diphenyl-2-
thioxo-pyrimidine-5-carboxamide (4).
Titled compound was synthesized following the general
procedure B (see SI) using compound II (100 mg, 0.27 mmol)
and 3-aminophenol (30 mg, 0.27 mmol) obtaining 29.6 mg of
pure compound (yield 26 %). Rt = 1.84 min; MS (SI) m/z:
ACKNOWLEDGMENT
This work was supported in part by the Italian Association
for Cancer Research (AIRC) through the “IG 18883” Grant
and by Università degli Studi di Padova (Grant # 60A04-
7255). We thank Grace Fox for her proofreading and copyedit-
ing
1
432.3 [M-H]-. [M-H]- calculated: 432.1. H NMR (400 MHz,
CDCl3) δ 11.84 (s, 1H), 7.68 – 7.43 (m, 6H), 7.33 (t, J = 6.9
Hz, 4H), 7.24 (t, J = 8.1 Hz, 1H), 7.13 (s, 1H), 6.98 (d, J = 8.0
Hz, 1H), 6.76 – 6.68 (m, 1H), 5.08 (s, 1H). 13C NMR (101
MHz, CDCl3) δ 178.29 (CS), 169.02 (CONH), 167.55 (C),
161.96 (C), 156.07 (C), 138.87 (C), 137.76 (C), 136.13 (C),
130.15 (CH), 129.56 (CH), 129.47 (CH), 129.09 (CH), 128.84
(CH), 128.43(CH), 128.31 (CH), 113.96 (CH), 113.24 (CH),
108.90 (CH), 83.38 (C). qNMR: 95.1 %.
ABBREVIATIONS
TopoII, type II topoisomerase; DNA, deoxyribonucleic acid;
DCM, dichloromethane; MeOH, methanol, EtOAC,
ethylacetate; DMF, N,N-dimethylformamide; DMAP, 4-(N,N-
dimethylamino)pyridine; Rt, retention time; SI, supporting in-
formation.
4-hydroxy-6-oxo-N,1,3-triphenyl-2-thioxo-pyrimidine-5-
carboxamide (5).
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pound (yield 86 %). Rt = 2.19 min; MS (SI) m/z: 414.1 [M-H]-
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