Design and synthesis of novel thiobarbituric acid derivatives targeting both wild-type and BRAF-mutated melanoma cells

Article abstract of DOI:10.1016/j.ejmech.2017.11.006

A series of novel thio- and seleno-barbituric acid derivatives were synthesized by varying the substituents at N1 and N3 (ethyl, methyl, allyl, and phenyl), and C5 tethered with dienyl and trienyl moieties attached to substituents such as phenyl, 2-furanyl, 2-thiophenyl, 1-naphthyl, and 3-pyridyl. The cytotoxic potential of these derivatives was evaluated by using MTT assay against melanoma cell lines expressing either wild-type (CHL-1) or mutant (UACC 903) BRAF gene. Among all, 2b and 8b were identified as the most potent compounds. Both 2b and 8b inhibited viability of various melanoma cells and induced cell death as evidenced by Live and Dead assay. Western blot analysis showed that they induce PARP cleavage and inhibit anti-apoptotic Bcl-2, Bcl-xL and Survivin in a dose-dependent manner within 24 h of the treatment. Novel thiobarbituric acid analogs also inhibited viability of various other solid tumor cell lines, such as pancreatic, breast, and colon. Overall, 2b, 2d, and 8b emerged as the most effective compounds and make good leads for the development of future therapeutic agents.

Full text of DOI:10.1016/j.ejmech.2017.11.006

Accepted Manuscript
Design and synthesis of novel thiobarbituric acid derivatives targeting both wild-type
and BRAF-mutated melanoma cells
Srinivasa Rao Ramisetti, Manoj K. Pandey, Sang Y. Lee, Deepkamal Karelia, Satya
Narayan, Shantu Amin, Arun K. Sharma
PII:
S0223-5234(17)30891-7
10.1016/j.ejmech.2017.11.006
EJMECH 9881
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 February 2017
Revised Date: 1 November 2017
Accepted Date: 2 November 2017
Please cite this article as: S.R. Ramisetti, M.K. Pandey, S.Y. Lee, D. Karelia, S. Narayan, S. Amin,
A.K. Sharma, Design and synthesis of novel thiobarbituric acid derivatives targeting both wild-type
and BRAF-mutated melanoma cells, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.11.006.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design and synthesis of novel thiobarbituric acid
derivatives targeting both wild-type and BRAF-
mutated melanoma cells
Srinivasa Rao Ramisetti,1 Manoj K Pandey,1,2 Sang Y. Lee,3 Deepkamal Karelia,1 Satya
Narayan,4 Shantu Amin,1 and Arun K. Sharma1,
1Department of Pharmacology; 3Department of Neurosurgery; Penn State Cancer Institute,
CH72; Penn State College of Medicine, 500 University Drive, Hershey, PA 17033, USA.
2Department of Biomedical Sciences, Cooper Medical School of Rowan University, 401 S
Broadway, Camden, NJ 08103 (current address)
4Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL 32610, USA
Author to whom correspondence should be addressed
Arun K. Sharma, Ph.D.
Department of Pharmacology
Penn State College of Medicine
Penn State Cancer Institute, CH72
Penn State Milton S. Hershey Medical Center
500 University Drive
Hershey, PA 17033
Phone: 717-531-4563
Fax:
717-531-0244
E-mail: aks14@psu.edu
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Abstract
A series of novel thio- and seleno-barbituric acid derivatives were synthesized by varying the
substituents at N1 and N3 (ethyl, methyl, allyl, and phenyl), and C5 tethered with dienyl and
trienyl moieties attached to substituents such as phenyl, 2-furanyl, 2-thiophenyl, 1-naphthyl, and
3-pyridyl. The cytotoxic potential of these derivatives was evaluated by using MTT assay against
melanoma cell lines expressing either wild-type (CHL-1) or mutant (UACC 903) BRAF gene.
Among all, 2b and 8b were identified as the most potent compounds. Both 2b and 8b inhibited
viability of various melanoma cells and induced cell death as evidenced by Live and Dead assay.
Western blot analysis showed that they induce PARP cleavage and inhibit anti-apoptotic Bcl-2,
Bcl-xL and Survivin in a dose-dependent manner within 24 h of the treatment. Novel
thiobarbituric acid analogs also inhibited viability of various other solid tumor cell lines, such as
pancreatic, breast, and colon. Overall, 2b, 2d, and 8b emerged as the most effective compounds
and make good leads for the development of future therapeutic agents.
Keywords: Thiobarbiturates, selenobarbiturates, anti-cancer, melanoma, apoptosis
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1. Introduction
Cancer is one of the leading causes of death worldwide and accounted for 8.2 million deaths
(nearly 14.6% of all deaths) in 2012.[1] Deaths from cancer worldwide are projected to continue
to rise to over 13 million annually within next two decades. In the United States alone, an
estimated 1,685,210 new cancer cases are diagnosed with estimated deaths of 595,690 in
2016.[2] Among all cancers, melanoma is one of the most deadly form of skin cancer with 2
million new cases annually in the United States.[2] If diagnosed and removed while it is still thin
and limited to the outermost skin layer, it is almost 100% curable. However, no effective therapy
is available for treatment once the cancer advances and metastasizes to other parts of the body.
Commonly used drugs, dacarbazine and temozolomide, in melanoma patients provide
unsatisfactory outcomes. The BRAFV600E inhibitor PLX4032 (vemurafenib)[3] (Figure 1) has
proved to be a remarkable drug but develops resistance in about 7 months, and also works only
on tumors with BRAFV600E mutations. Most patients require multiple lines of therapy as cancer
cells acquire resistance against the chemotherapeutic agents by different mechanisms. The
combination therapies with BRAF and MEK inhibitors such as Cobimetinib or Trametinib
(Figure 1) also did not show much improvement.[4] Management of patients with resistance
continues to present an extensive confront to researchers and oncologists. Many clinically
effective anti-cancer drugs, including those for melanoma used today, suffer from serious
drawbacks as majority of patients with metastatic solid tumors die after chemotherapeutic
regimens failed to induce objective response or when resistance developed.[5] Therefore, to
combat this resistance, still there is an impetus to identify and develop more potent therapeutic
agents with fewer side effects in cancer therapy.
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In recent years, thiobarbituric acids have been reported to exhibit a broad spectrum of
biological properties such as anti-convulsant,[6] anti-inflammatory,[7, 8] anti-bacterial,[9] anti-
HIV,[10] anti-hypnotic,[11] anti-parkinsonian,[12] and anti-cancer activities,[13-15] including
anti-melanoma properties.[16, 17] Among them some thiobarbituric acid derivatives, such as
merbarone,[18] merocyanine 540[19] and its selenium analog selenomerocyanine 56[20] (Figure
1) have been identified as possible modulators of apoptosis in several cancer cells, including
glioblastoma multiforme (GBM), melanoma, leukemia, and cancers of breast, lung, prostate, and
cervix; albeit not particularly for cancers that are resistant to current therapy. An effective lead
thiobaribituic acid analog 2a was recently identified in our laboratory from a library of 50,000
chemicals, originally screened to identify compounds that could target temozolomide (TMZ)
resistant brain tumor cells. The lead compound 2a was effective on both TMZ resistant and
sensitive glioma cells.[21] Interestingly, our preliminary studies showed 2a to also possess
activity towards human melanoma cells. Therefore, we hypothesized that the structure of 2a can
be modulated by varying substitutions at N1, N3, and C5 positions to create a potent compound
for melanoma treatment. Therefore, in the present study, we conducted a structure-activity
relationship (SAR) study to further optimize 2a structure to develop an efficacious novel thio-
/seleno-barbituric acid compound for melanoma and possibly other solid tumors. In addition, 2a
was considered as a valuable lead compound owing to its selective cytotoxicity to various cancer
cells including melanoma, while sparing the normal cells. We synthesized 24 novel thio- and
selenobarbituric derivatives by varying the substituents at N1, N3, and C5 tethered with dienyl
and trienyl moieties attached with different substituents like phenyl, 2-furanyl, 2-thiophenyl, 1-
naphthyl and 3-pyridyl. The efficacy of novel analogs was evaluated in a diverse panel of cancer
cell lines and the two best identified compounds 2b and 8b, were further characterized in both
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wild-type and BRAFV600E mutant melanoma cells to get an insight into their mechanism of
action.
2. Results and discussion
2.1. Design
The lead thiobarbituric acid analog 2a was found to be effective on both BRAF-mutant and wild-
type melanoma cells. We hypothesized that careful manipulation of 2a could lead to more
effective analogs for a broader range of melanomas as well as other cancers. In this regard, we
modified various functionalities by manipulating substitutions at N1- , N3- and C5-positions of
2a as well as by replacing S by Se (Figure 1). While choosing substitutions, we also made sure
that the resulting modified compounds satisfy structural parameters of Lipinski’s Rule-of-
five[22, 23] for drug-likeness.
Our first focus was to modify 3-furan-2-yl allylidene functionality at C5 position of 2a.
The modifications included replacing terminal furan ring with phenyl, thiophenyl, and naphthyl
substitutions and extending the linker carbon chain length from dienyl to trienyl unit.
Compounds 2b-f were thus synthesized to determine the effect of chain length and terminal
aromatic ring at C5 position of 2a. Several studies have pointed out the anti-tumor and chemo-
preventive effects of multiple Se species in various cancers.[24-29] Recent reports by our
laboratories and other groups have shown that replacement of S by Se in naturally occurring and
synthetic isothiocyanates and several other compounds, in general though not always,
substantially enhanced the efficacy of resulting compounds against melanoma and several other
cancers.[28-32] Therefore, our second modification strategy involved replacing S in
thiobarbituric acid ring by Se. We synthesized selenobarbituric acid analogs of 2a and two other
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effective thio-analogs 2b and 2c, to evaluate if the replacing S by Se in such scaffold would help
enhance the cytotoxicity.
Finally, to determine the effect of N-substitutions on thiobarbituric acid compounds 2, we
generated a series of N1-monosubstituted, N1,N3-disubstituted analogs 8a-o. The substitutions at
N1 and N3 included methyl, ethyl, allyl, and phenyl groups. Overall, all the N1-substituted, N1,
N3-disubstituted, and N1- and N3-unsubstituted derivatives were synthesized to study the effect
of partially substituted, fully substituted, and free NH compounds with varying functionalities at
C5 position.
2.2. Chemistry
Novel thio- (2 and 8) and seleno- (5) barbituric acid derivatives were prepared according to the
sequence of reactions depicted in Schemes 1‒3. The thiobarbituric acid analogs 2a-f were
synthesized by Knoevenagel condensation of readily available thiobarbituric acid 1 with
different α,β-unsaturated aldehydes in the presence of catalytic amount of pyridine in EtOH
under reflux condition (Scheme 1).[33] To evaluate the difference in activity between the S
compounds 2 and corresponding Se analogs, we synthesized isosteric selenium analogs of lead
compound 2a and other key thiobarbituric acid analogs 2b, c. The key intermediate
selenobarbituric acid 4 was synthesized in good yield by treating readily available selenourea 3
with diethylmalonate in the presence of freshly prepared sodium ethoxide in ethanol under reflux
conditions.[34] Subsequently the selenobarbituric acid 4 was reacted under Knoevenagel
condensation with different aldehydes in the presence of catalytic pyridine in refluxing EtOH to
furnish the corresponding novel selenobarbituric acid derivatives 5a-c in good to excellent yields
(Scheme 2).[33]
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To further diversify the structure activity relationship study on thiobarbitric acid
derivatives, we further synthesized N1-monosubstituted and N1,N3-disubstituted thiobarbituric
acid derivatives 8a-o. The precursor substituted thiobarbituric acid intermediates 7a-g were first
synthesized by condensing appropriately substituted thioureas 6a-e with diethyl malonate in the
presence of freshly prepared sodium ethoxide in refluxing ethanol.[34] The substituted
thiobarbituric acids 7a-g were then reacted with different α,β-unsaturated aldehydes under
Knoevenagel condensation to furnish the corresponding novel N1-monosubstituted and N1,N3-
disubstituted thiobarbituric acid derivatives 8a-o in good to excellent yields (Scheme 2).[33] The
1
structures of all the novel thio- and seleno-barbituric acid derivatives were confirmed by H
NMR, 13C NMR and HRMS analysis. The compounds purity (≥98%) was analyzed by analytical
high-performance liquid chromatography (HPLC) before proceeding for in vitro biological
assays.
2.3. Biology
2.3.1. Evaluation of anti-proliferative activities of novel compounds against CHL-1 (BRAF
wild-type) and UACC903 (BRAF-mutant) melanoma cell lines
The anti-proliferative activities of thio- and seleno-barbituric acid derivatives (2, 5, and 8) were
evaluated against both BRAF-wild-type (CHL-1) and BRAF-mutant (UACC 903) melanoma cell
lines. The cells were treated with various compounds for 72 h and the cell survival was
determined by utilizing MTT assay. Among N1- and N3-unsubstituted compounds 2a-f,
compounds 2b and 2d where furanyl ring of 2a was replaced by thiophenyl and naphthyl groups,
respectively, were more effective in inhibiting viability of both CHL-1 and UACC903 cells as
compared to parent compound 2a (Table 1). The phenyl or pyridyl group substitutions
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(compounds 2c and 2e, respectively) led to decrease in potency, 2e being the least effective.
Interestingly, changing the chain from dienyl to trienyl with terminal phenyl ring (compound 2f)
again restored to potency of the molecule. Notably, similar to as observed with lead compound
2a, all the new compounds 2b-f showed similar cell viability inhibition against both BRAF wild-
type and mutant cells.
Previous studies, both from our group[28-32] and others,[35] have shown isosteric
replacement of sulfur by selenium to enhance the cytotoxicity of small molecules. Examples
include increase in potency from isothiocyantes to isoselenocyantes,[30] isothioureas to
isoselenoureas[28] among others, although it is not always the case.[36] Interestingly, in the
present case, replacement of S in thiobarbituric acid ring of 2a by Se, led to lowering of potency
of the resulting selenobarbitutic acid analog 5a (Table 1). Similarly, selenothiobarbituric acids
5b and 5c were less effective than their sulfur isosteres 2b and 2c; except that 5c showed a
similar IC50 value as 2c. This indicates that presence of selenium does not necessarily increase
the toxicity of a molecule but the actual effect would depend on overall molecular structure and
position of the Se atom.
The N1-monosubstituted and N1,N3-disubstituted thiobarbituric acid analogs (8a-o)
demonstrated variable efficacy depending on the combination of type of N-substitution(s) and C-
5 moiety. Although no clear structure-activity relationship could be derived, it was observed that
8d derived by incorporating an ethyl group at N1-position of 2a resulted in loss of activity while
N1,N3-diethyl analog 8j retained activity against BRAF wild-type CHL-1 cells while lost
cytotoxicity against BRAF-mutated UACC903 cells. Extension of chain length at C5 from dienyl
to tienyl linker (compound 8h) again restored the activity (IC50 ~7.5 µM). Similar to N1- and
N3-unsubstituted compound 2b (Table 1), the compounds 8a, 8b, 8c, 8e, and 8m (Table 2) with
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thiophenyl terminal ring at C5 position were potent irrespective N1-monosubstitution as allyl,
phenyl, methyl, ethyl or N1-allyl-N3-ethyl disubstitution. This suggested that 2-thiophenyl ring
at terminal position of C5 substitution is essentially responsible for the activity which is retained
irrespective of substitutions at N1 and N3. The combination of naphthyl group at C5 and N-ethyl
(compound 8g) led to a substantial loss of activity (IC50 ~23 µM). N1-allyl-N3-ethyl
disubstitution in combination with furanyl (5k) or phenyl (5l) also led to decrease in potency.
Interestingly, compound 5j (N1-ethyl and phenyl at C5) and 5n and 5o (N1-allyl and furanyl and
phenyl at C5, respectively) resulted in selective cytotoxicity towards CHL-1 cells.
Overall, 2b, 2d, 2f, 5b (Table 1), 8a, 8b, 8c, 8e, 8f, 8h, and 8m (Table 2) emerged as
most potent anti-proliferative compounds with IC50 lower than lead compound 2a and non-
specifically targeted both UACC903 and CHL-1 cells. Among these compounds 2b and 8b were
the most effective and were chosen for further studies. Importantly, unlike PLX4032, compound
2b and 8b were found equally potent in killing melanoma cells irrespective of mutation status
(Table 2). As shown in Tables 1 and 2, compounds 2b and 8b inhibited growth of both CHL-1
and UACC 903 cell lines-. The fact that these compounds were equally effective in both BRAF-
mutant and wild-type melanoma cells, suggest that the inhibitory activities of compounds 2b and
8b are independent of BRAF expression. Therefore, we extended our investigation to elucidate
their mechanism of cell death and cell growth inhibition.
2.3.2. Compound 2b and 8b inhibited viability of a variety of melanoma cells
Based on the results of cell viability inhibition after 72 h treatment by the novel compounds
against UACC903 and CHL-1 cell lines (Tables 1 and 2), the two best identified compounds 2b
and 8b were selected for further screening against five human melanoma cell lines at 48 h time
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point. Both compounds 2b and 8b were essentially equally effective in inhibiting viability of
various melanoma cells. The IC50 values for CHL-1 and UACC 903 cells at 48 h treatment were
in the range of 6.5-8.1 µM (Table 3) as compared to 3.8-4.0 µM (Tables 1 and 2) for 72 h
treatment. Both the compounds showed a similar potency against WM2664 cells. The
compounds were slightly less effective against 1205Lu and A375M cell lines (IC50 between 10.6
and 14.8 µM, Table 3), compound 2b being slightly more potent. Figure 3 shows the cell
viability curves for compounds 2b and 8b in all five melanoma cell lines at 48 h time point.
2.3.3. Compound 2b and 8b cause cell death by inducing apoptosis
Decrease of cell viability seen in MTT assay can be attributed to cell growth inhibition and/or
cell death. We further confirmed our MTT assay observation by using another technique
popularly known as Live/Dead assay. The Live/Dead assay determines intracellular esterase
activity and plasma membrane integrity. UACC903 and CHL-1 cells were treated with 2b and
8b for 24 h. As shown in Figure 4, treatment of melanoma cells with 2b and 8b increase the
number of dead cells, which reflects the cytotoxic potential of these analogs in melanoma cells.
Compound 2b was more effective, in both CHL-1 and UACC903 cells, as compared to 8b.
However, results from this assay suggest that 8b may potentially inhibit cell growth and induce
cell death later, since it showed less death at 25 µM (25% in UACC903; 5% in CHL-1), while in
MTT assay it was even more potent at reducing the cell viability for both the cell lines. Our data
indicates 2b to be relatively more effective at inducing cell death than 8b.
We further examined the effects of 2b and 8b on cell death using several other techniques
such as Annexin V assay and measuring the activation of caspase 3/7. UACC903 cells were
treated with given concentrations of 2b and 8b for 24 h. As shown in Figure 5A, vehicle-treated
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cells were mostly present in the bottom left quadrant. However, as the dose of 2b and 8b was
escalated, cells shifted from live to early apoptotic and towards late apoptotic dead cells. Total
apoptotic death obtained by Annexin V and Caspase 3/7 activity assays was similar to our
Live/Dead assay. More than 70% cells were positive for both Annexin V, Caspase 3/7 activity
and 7-AAD, indicating apoptotic dependent cell death. Further, necrotic death, cells only positive
for 7-AAD and negative for Annexin V and Caspase 3/7 activity (cells present in the top left
quadrant), was not detected. Overall, our results confirm that 2b and 8b inhibits the growth of
melanoma cells by inducing apoptosis, indicated by presence of phosphatidylserine (PS) on outer
membrane and positive for caspase 3/7 activity.
2.3.4. Compound 2b and 8b inhibited the expression of survival proteins in melanoma
Escaping from cell death is one of the adaptations that enable cancer cells to stave off anticancer
therapies. The key players in avoiding apoptosis are collectively known as survival proteins.
Survival proteins mainly comprise the Bcl-2 and inhibitor of apoptosis (IAP) families.[37] The
aberrant expression of these proteins is associated with a range of biological activities that
promote cancer cell survival, proliferation, and resistance to therapy.[37] We determined the
effect of compounds 2b and 8b on the expression of survival proteins. As shown in the Figure 6,
compound 2b and 8b showed differential response on survival proteins. Treatment of these
analogs induces phosphorylation of Bcl-2, which is required for its degradation, inhibits
expression of anti-apoptotic proteins like Bcl-xL, Survivin and finally activates PARP cleavage.
Thus, our results further support that these analogs have potential to be used as therapeutic
agents.
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2.3.5. Novel thiobarbituric acid analogs were also effective against cancers other than
melanoma and were more selective towards cancer cells.
To evaluate the effect of novel thiobarbituric acid analogs on cancers other than melanoma, a
select effective compounds (2b, 2d, 8b, 8c, and 8e) identified from melanoma cells screen were
tested against a panel of solid cancer cell lines, e.g., Panc-1 (Pancreatic cancer), MDA-MB-231
(ER/PR/HER2 negative breast cancer), MCF-7 (ER/PR/HER2 positive breast cancer), and
HCT116 (colon cancer). Interestingly, compound 2d was most effective against all the cell lines
tested with IC50s in the range of 3.6-7.9 µM (Table 4 and Figures 7A and B). All the compounds
tested were effective against MDA-MB-231 cells with single digit IC50 values as compared to the
lead compound 2a (IC50 ~16.5 µM). Compound 2b selectively inhibited viability of MDA-MB-
231 and HCT-116 cells while compound 8b was selective towards Panc-1, MDA-MB-231, and
HCT116 cells, being most effective against HCT116 cells (IC50 ~7.7 µM). Figure 3 shows the
cell viability curves for compounds 2b and 8b in all five melanoma cell lines at 48 h time point.
The cell viability curves for compounds 2b and 8b in HCT116, Panc-1, MDA-MB-231, and
MCF-7 cell lines at 48 h time point are shown in Figures 7A and B. These data show that
compounds 2b and 8b potently inhibited viability of most, although not all, of the tested cancer
cell lines, suggesting that these compounds could be widely used against cancer cells.
To get an insight into the selectivity of these compounds towards cancer cells, we
performed MTT cell viability assay at the highest concentration (25 µM) of both the compounds
(2b and 8b) in normal cell line MCF10A, the human breast epithelial cell line. After 48 h
treatment, the normal cell line showed ~60% cell viability while the cancer cell lines exhibited
~20%-30% cell viability (Figures 7C and D). Both the compounds were about two-fold more
potent at killing breast cancer cells (MCF-7 and MDA-MB-231) and three-fold more potent
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towards melanoma cells (UACC903) as compared to the normal MCF10A cells. These data
indicate that both the agents have some selectivity towards cancer cells than normal cells.
However, in vitro studies are often not a true measure of toxicity/efficacy balance and therefore
the future in vivo efficacy and toxicity studies would reveal the real therapeutic window of these
compounds. Furthermore, the mechanism behind selective targeting of certain cancer cell types
remains to be elucidated.
3. Conclusion
We synthesized a series of thio- and seleno-barbituric acid analogs with a variety of substitutions
at N1, N3, and C5 positions. Many of these compounds inhibited viability of both BRAF wild-
type (CHL-1) and mutant (UACC903) human melanoma cells. Based on the SAR study
compounds 2b, 2d, and 8b were found to be the most effective analogs. Live/Dead assay showed
compound 2b to be more effective than 8b in inducing cell death; causing nearly 65-70% cell
death at 25 µM concentration in human melanoma cells. Both compounds 2b and 8b effectively
induced apoptosis in both CHL-1 and UACC903 cell lines. The western blot analysis showed
that 2b and 8b induced PARP cleavage and reduced the levels of anti-apoptotic proteins Bcl-2,
Bcl-xL and Survivin in a dose-dependent manner. Further extensive studies are, however,
required to reveal the mechanism of these novel thiobarbituric acid derivatives. Many of the
novel thiobarbituric acid analogs were also effective in inhibiting viability of various other solid
cancer cell lines, e.g., pancreatic, breast, and colon demonstrating that these compounds possess
anti-proliferative ability against variety of cancer cells. Notably, compounds 2b and 8b inhibited
growth of melanoma cells irrespective of mutational status of BRAF. Owing to the fact that the
new compounds act independently of BRAF mechanism, they might potentially overcome the
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issue of resistance to currently clinically used BRAF-inhibitor PLX4032 that selectively targets
BRAF-mutant tumors. Further pre-clinical studies including in vivo efficacy, toxicity, and
mechanism of action, are, however, necessary to determine the future clinical potential of these
compounds.
4. Experimental
4.1. Chemistry
4.1.1. General
Melting points were recorded on a Fischer-Johns melting point apparatus and are uncorrected.
NMR spectra were recorded using a Bruker Avance 500 MHz spectrometer. Chemical shifts (δ)
are reported in parts per million (ppm) downfield from the internal standard. The signals are
quoted as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (doublet of doublet) and
dt (doublet of triplet). HRMS were determined at the Chemistry Instrumentation Center, State
University of New York at Buffalo, NY. Thin-layer chromatography (TLC) was developed on
aluminum-supported pre-coated silica gel plates (EM industries, Gibbstown, NJ). Column
chromatography was conducted on silica gel (60-200 mesh). The substituted thioureas, e.g., N,N-
diallyl- (6a), N,N-diethyl- (6b), N-allyl-N′-ethyl- (6c) and N-ethyl- (6d) thioureas were obtained
from commercial sources.
4.1.2. General procedure for the synthesis of thiobarbiturates (2a-f)
To a stirred solution of 1,3-thio-barbituric acid (1) (1.0 mmol), and corresponding trans-α,β-
unsaturated aldehydes (1.0 mmol) in absolute ethyl alcohol (5 mL) under nitrogen atmosphere,
was added dry pyridine (0.2 mmol) and the resulting mixture was allowed to stir at reflux for 12
h. After completion of the reaction as indicated by TLC, the solvent was removed under reduced
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pressure. The residue thus obtained was washed with hot H2O (2 × 10 mL), cold EtOH (2 × 5
mL) and Et2O (2 × 10 mL) to afford crude thiobarbiturates (2a-f), which further recrystallized
from 1,4-dioxane-water to afford pure compounds.
4.1.3. 5-(3-Furan-2-yl-allylidene)-2-thioxo-dihydro-pyrimidine-4,6-dione (2a)
Yield 87%; reddish solid; mp 245 oC (dec.); 1H NMR (500 MHz, DMSO-d6) δ [ppm]: 8.20 (dd, J
= 12.5, 10.5 Hz, 1H), 8.03–8.00 (m, 2H), 7.62 (d, J = 12.5 Hz, 1H), 7.08 (d, J = 3.0 Hz, 1H),
13
6.75–6.73 (m, 1H); C NMR (125 MHz, DMSO-d6) δ [ppm]: 178.84, 161.86, 161.42, 154.80,
152.35, 148.58, 139.40, 122.78, 119.65, 115.43, 114.44; HRMS calcd for C11H8N2O3S:
248.0250. Found 248.0256.
4.1.4. 5-(3-Thiophen-2-yl-allylidene)-2-thioxo-dihydro-pyrimidine-4,6-dione (2b)
o
1
Yield 82%; brown solid; mp 270 C (dec.); H NMR (500 MHz, DMSO-d6) δ [ppm]: 12.29,
12.25 (2s, 2 × NH), 8.24–8.14 (m, 1H), 8.06–7.76 (m, 3H), 7.55 (s, 1H), 7.25 (s, 1H); 13C NMR
(125 MHz, DMSO-d6) δ [ppm]: 178.90, 161.84, 161.43, 154.92, 146.82, 141.48, 134.61, 133.35,
129.94, 124.21, 115.27; HRMS calcd for C11H8 N2O2S2: 264.0022. Found 264.0028.
4.1.5. 5-(3-Phenyl-allylidene)-2-thioxo-dihydro-pyrimidine-4,6-dione (2c)
o
1
Yield 89%; yellow solid; mp 265 C; H NMR (500 MHz, DMSO-d6) δ [ppm]: 8.46 (dd, J =
13.0, 10.0 Hz, 1H), 8.06 (d, J = 9.5 Hz, 1H), 7.80 (d, J = 13.0 Hz, 1H), 7.68-7.72 (m, 2H), 7.48-
7.53 (m, 3H); 13C NMR (125 MHz, DMSO-d6) δ [ppm]: 179.04, 161.81, 161.31, 155.11, 154.18,
135.78, 131.96, 129.81, 129.27, 125.07, 116.44; HRMS calcd for C13H10N2O2S: 258.0457.
Found 258.0461.
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4.1.6. 5-(3-Naphthalen-1-yl-allylidene)-2-thioxo-dihydro-pyrimidine-4,6-dione (2d)
o
1
Yield 78%; reddish brown solid; mp 247 C; H NMR (500 MHz, DMSO-d6) δ [ppm]: 12.36,
12.32 (2s, 2 × NH), 8.77 (d, J = 15.0, 1H), 8.61 (dd, J = 15.5, 12.0 Hz, 1H), 8.50 (d, J = 8.5 Hz,
13
1H), 8.33 (d, J = 12.0 Hz, 1H), 8.00-8.10 (m, 3H), 7.70-7.60 (m, 3H); C NMR (125 MHz,
DMSO-d6) δ [ppm]: 179.03, 161.91, 161.38, 155.50, 150.47, 134.00, 132.47, 132.41, 131.67,
129.32, 127.91, 127.09, 127.03, 126.41, 126.24, 123.80, 116.36; HRMS calcd for C17H12N2O2S:
308.0614. Found 308.0618.
4.1.7. 5-(3-Pyridin-3-yl-allylidene)-2-thioxo-dihydro-pyrimidine-4,6-dione (2e)
o
1
Yield 72%; yellow solid; mp 274 C (dec.); H NMR (500 MHz, DMSO-d6) δ [ppm]: 12.37,
12.34 (2s, 2 × NH), 8.82 (d, J = 2.0 Hz, 1H), 8.62 (dd, J = 4.5, 1.5 Hz, 1H), 8.48 (dd, J = 16.0,
12.0 Hz, 1H), 8.14 (dt, J = 8.0, 2.0 Hz, 1H), 8.04 (d, J = 11.5 Hz, 1H), 7.80 (d, J = 15.5 Hz, 1H),
7.52 (dd, J = 8.0, 5.0 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ [ppm]: 179.10, 161.70, 161.17,
154.04, 151.86, 150.52, 149.87, 135.25, 131.64, 126.63, 124.87, 117.40; HRMS calcd for
C12H9N3O2S: 259.0410. Found 259.0407.
4.1.8. 5-(5-Phenyl-penta-2,4-dienylidene)-2-thioxo-dihydro-pyrimidine-4,6-dione (2f)
o
1
Yield 75%; reddish brown solid; mp 241 C; H NMR (500 MHz, DMSO-d6) δ [ppm]: 12.29,
12.23 (2s, 2 × NH), 8.02– 7.86 (m, 2H), 7.68 (d, J = 7.0 Hz, 2H), 7.58 (dt, J = 11.0, 2.0 Hz, 1H),
7.42-7.38 (m, 4H), 7.68 (d, J = 15.5 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ [ppm]: 178.85,
161.92, 161.30, 155.63, 154.99, 144.03, 136.39, 130.34, 129.48, 129.45, 129.39, 128.44, 115.28;
HRMS calcd for C15H12N2O2S: 284.0614. Found 284.0613.
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4.1.9. Synthesis of selenobarbituric acid analogs (5a-c)
The key intermediate 2-selenobarbarbituric acid (4) was prepared by refluxing selenourea (3)
with diethylmalonate in ethanol in the presence of sodium methoxide by modification of a
previously reported method.[38] Briefly, to a stirred solution of sodium ethylate [freshly
prepared by dissolving sodium (0.373 g, 16.24 mmol) in absolute ethanol (10 mL)] under
nitrogen atmosphere was added diethyl malonate (2.59 g, 16.24 mmol) followed by selenourea
(3) (0.50 g, 4.06 mmol) and the resulting mixture was allowed to stir at reflux for 45 min. After
completion of the reaction as indicated by TLC, the reaction mixture was chilled and the
resulting white solid was filtered. The second crop of solid was obtained by adding Et2O (10 mL)
to the filtrate. The combined solids were dissolved in ice-cold water (10 mL) and the solution
was acidified with dilute HCl. The resulting solid was filtered, washed with cold water, Et2O and
dried to afford 2-selenoxo-dihydro-pyrimidine-4,6-dione (4) (mp dec. >200 oC), which was pure
enough to carry to the next step. The seleno-barbiturates (5a-c) were synthesized by treating 4
(1.0 mmol) with trans-α,β-unsaturated aldehydes (1.0 mmol) following the same reaction, work-
up, and purification sequence as described above for compound 2a-f.
4.1.10. 5-(3-Furan-2-yl-allylidene)-2-selenoxo-dihydro-pyrimidine-4,6-dione (5a)
o
1
Yield 77%; brown solid; mp 277 C (dec.); H NMR (500 MHz, DMSO-d6) δ [ppm]: 12.70,
12.66 (2s, 2 × NH), 8.18 (dd, J = 14.5, 12.0 Hz, 1H), 8.10–8.05 (m, 2H), 7.70 (d, J = 15.0 Hz,
1H), 7.14 (d, J = 4.0 Hz, 1H), 6.76 (dd, J = 4.0, 3.5 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ
[ppm]: 179.80, 161.33, 160.79, 155.28, 152.47, 148.79, 139.69, 123.03, 119.99, 115.71, 114.59;
HRMS calcd for C11H8N2O3Se: 295.9695. Found 295.9701.
17

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4.1.11. 2-Selenoxo-5-(3-thiophen-2-yl-allylidene)-dihydro-pyrimidine-4,6-dione (5b)
Yield 81%; brown solid; mp 275–277 oC (dec.); 1H NMR (500 MHz, DMSO-d6) δ [ppm]: 12.71,
12.66 (2s, 2 × NH), 8.18–8.12 (m, 1H), 8.10–8.04 (m, 2H), 7.96 (d, J = 11.0 Hz, 1H), 7.62 (d, J
13
= 3.5 Hz, 1H), 7.27–7.23 (m, 1H); C NMR (125 MHz, DMSO-d6) δ [ppm]: 179.85, 161.30,
160.79, 155.42, 147.18, 141.59, 134.82, 133.63, 130.05, 124.46, 115.56; HRMS calcd for
C11H8N2O2SSe: 311.9466. Found 311.9478.
4.1.12. 5-(3-Phenyl-allylidene)-2-selenoxo-dihydro-pyrimidine-4,6-dione (5c)
o
1
Yield 85%; reddish brown solid; mp 270–272 C; H NMR (500 MHz, DMSO-d6) δ [ppm]:
12.71, 12.74 (2s, 2 × NH), 8.42 (dd, J = 15.5, 12.0 Hz, 1H), 8.23 (d, J = 12.0 Hz, 1H), 7.86 (d, J
= 15.5 Hz, 1H), 7.68–7.72 (m, 2H), 7.48–7.52 (m, 3H); 13C NMR (125 MHz, DMSO-d6) δ
[ppm]: 180.14, 161.27, 160.67, 155.58, 154.51, 135.85, 132.06, 129.86, 129.32, 125.33, 116.77;
HRMS calcd for C13H10N2O2Se: 305.9902. Found 305.9899.
4.1.13. General procedure for the synthesis of thiobarbiturates (8a-o)
To a stirred solution of sodium ethylate [freshly prepared by dissolving sodium (4.0 mmol) in
absolute ethyl alcohol (10 mL)] under nitrogen atmosphere was added diethyl malonate (4.0
mmol) followed by substituted thioureas 6a-d (1.0 mmol) and the resulting mixture was allowed
to stir at reflux for 48 h. After completion of the reaction as indicated by TLC, the reaction
mixture was diluted with H2O (10 mL). Then most of the ethyl alcohol was removed under
reduced pressure. The residue was poured into cold water (10 mL), chilled and filtered. The
aqueous layer was washed with Et2O (2 × 10 mL) to remove any unreacted diethyl malonate.
18

ACCEPTED MANUSCRIPT
The aqueous layer was acidified with dilute HCl and resulting solid was filtered off, washed with
cold water and Et2O, and dried to afford thiobarbituric acids (8a-o). Compounds 8a-h were
purified by crystallization from ethyl alcohol. The N1,N3-disubstituted compounds 8i-o were
purified by flash column chromatography using silica gel (100–200 mesh) eluting with n-
hexane/EtOAc (98:2) to afford the corresponding pure thiobarbiturates.
4.1.14. 1-Allyl-5-(3-thiophen-2-yl-allylidene)-2-thioxo-dihydro-pyrimidine-4,6-dione (8a)
Yield 82% (E & Z isomers); orange solid; mp 221 oC; 1H NMR (500 MHz, DMSO-d6) δ [ppm]:
12.50, 12.45 (2s, 2 × NH), 8.24–8.14 (m, 1H), 8.10–8.00 (m, 2H), 7.94 (t, J = 4.0 Hz, 1H), 7.60
(t, J = 4.0 Hz, 1H), 7.27–7.23 (m, 1H), 5.99–5.79 (m, 1H), 5.20–5.10 (m, 2H), 4.91–4.87 (m,
2H); 13C NMR (125 MHz, DMSO-d6) δ [ppm]: 179.36, 179.26, 161.44, 160.77, 160.28, 159.95,
156.51, 155.94, 147.72, 147.31, 141.47, 134.96, 134.88, 133.75, 133.63, 132.52, 132.43, 130.02,
124.39, 124.10, 117.33, 115.28, 115.11, 48.23, 47.90; HRMS calcd for C14H12N2O2S: 304.0335.
Found 304.0345.
4.1.15. 1-Phenyl-5-(3-thiophen-2-yl-allylidene)-2-thioxo-dihydro-pyrimidine-4,6-dione (8b)
Yield 90% (E & Z isomers); orange solid; mp 287 oC; 1H NMR (500 MHz, DMSO-d6) δ [ppm]:
12.62, 12.59 (2s, 2 × NH), 8.26 (dd, J = 12.5, 10.5 Hz, 1H), 8.13–8.08 (m, 2H), 8.06–8.00 (m,
3H), 7.92 (dd, J = 14.5, 4.0 Hz, 2H), 7.60–7.56 (m, 2H), 7.47–7.42 (m, 4H), 7.40–7.36 (m, 2H),
13
7.28–7.20 (m, 6H); C NMR (125 MHz, DMSO-d6) δ [ppm]: 180.13, 180.03, 162.09, 161.41,
160.78, 160.45, 155.89, 155.47, 147.34, 147.11, 141.49, 139.68, 139.47, 134.81, 134.79, 133.62,
133.52, 129.98, 129.69, 129.59, 129.22, 128.61, 124.26, 124.11, 115.72, 115.55; HRMS calcd
for C17H12N2O2S2: 340.0335. Found 340.03351.
19

ACCEPTED MANUSCRIPT
4.1.16. 1-Methyl-5-(3-thiophen-2-yl-allylidene)-2-thioxo-dihydro-pyrimidine-4,6-dione (8c)
1
Yield 89% (E & Z isomers); orange solid; mp 245oC (dec.); H NMR (500 MHz, DMSO-d6) δ
[ppm]: 12.45, 12.40 (2s, 2 × NH), 8.24–8.18 (m, 1H), 8.08–8.01 (m, 2H), 7.93 (t, J = 4.0 Hz,
1H), 7.59 (t, J = 4.0 Hz, 1H), 7.26–7.23 (m, 1H), 3.50 (s, 3H); 13C NMR (125 MHz, DMSO-d6) δ
[ppm]: 179.84, 179.75, 162.00, 161.33, 160.33, 160.01, 156.34, 155.63, 147.44, 147.15, 141.50,
141.46, 134.84, 134.79, 133.60, 133.54, 130.00, 129.97, 124.39, 124.13, 115.36, 115.25, 33.91,
33.50; HRMS calcd for C12H10N2O2S2: 278.0178. Found 278.0184.
4.1.17. 1-Ethyl-5-(3-furan-2-yl-allylidene)-2-thioxo-dihydro-pyrimidine-4,6-dione (8d)
Yield 85% (E & Z isomers); reddish brown solid; mp 245 oC; 1H NMR (500 MHz, DMSO-d6) δ
[ppm]: 12.38, 12.35 (2s, 2 × NH), 8.25 (dd, J = 15.0, 12.5 Hz, 1H), 8.10–8.02 (m, 2H), 7.66 (dd,
J = 15.0, 9.5 Hz, 1H), 7.12 (dd, J = 8.5, 3.5 Hz, 1H), 6.80–6.70 (m, 1H), 4.34–4.28 (m, 2H), 1.18
13
(q, J = 7.0 Hz, 3H); C NMR (125 MHz, DMSO-d6) δ [ppm]: 179.13, 179.04, 161.43, 160.74,
160.29, 159.93, 156.16, 155.50, 152.41, 152.35, 148.76, 148.71, 139.97, 139.65, 122.98, 122.79,
119.92, 119.89, 115.63, 115.50, 114.53, 114.50, 41.63, 41.27, 12.75, 12.72; HRMS calcd for
C13H12N2O3S: 276.0563. Found 276.0564.
4.1.18. 1-Ethyl-5-(3-thiophen-2-yl-allylidene)-2-thioxo-dihydro-pyrimidine-4,6-dione (8e)
o
Yield 87% (E & Z isomers); reddish solid; mp 272 C (dec.); 1H NMR (500 MHz, DMSO-d6) δ
[ppm]: 12.41, 12.37 (2s, 2 × NH), 8.24–8.18 (m, 1H), 8.10–8.00 (m, 2H), 7.95–7.90 (m, 1H),
13
7.62–7.58 (m, 1H), 7.30–7.20 (m, 1H), 4.34–4.28 (m, 2H), 1.18 (q, J = 7.0 Hz, 3H); C NMR
(125 MHz, DMSO-d6) δ [ppm]: 179.15, 179.09, 161.41, 160.79, 160.26, 159.92, 156.31, 155.70,
20

ACCEPTED MANUSCRIPT
147.52, 147.13, 141.50, 141.47, 134.86, 134.79, 133.64, 133.53, 129.99, 129.96, 124.40, 124.16,
115.45, 115.30, 41.64, 41.28, 12.77, 12.73; HRMS calcd for C13H12N2O2S2: 292.0335. Found
292.0331.
4.1.19. 1-Ethyl-5-(3-phenyl-allylidene)-2-thioxo-dihydro-pyrimidine-4,6-dione (8f)
Yield 92% (E & Z isomers); yellow solid; mp 256 oC; 1H NMR (500 MHz, DMSO-d6) δ [ppm]:
12.45, 12.41 (2s, 2 × NH), 8.50–8.45 (m, 1H), 8.10 (dd, J = 11.5, 4.0 Hz, 1H), 7.82 (dd, J =
15.5, 10.5 Hz, 1H), 7.72–7.68 (m, 2H), 7.52–7.48 (m, 3H), 4.32–4.28 (m, 2H), 1.18 (q, J = 7.0
Hz, 3H); 13C NMR (125 MHz, DMSO-d6) δ [ppm]: 179.32, 179.24, 160.71, 160.26, 159.84,
156.45, 155.86, 154.89, 154.46, 135.77, 132.10, 132.06, 129.83, 129.40, 129.34, 125.27, 125.05,
116.71, 116.54, 41.69, 41.34, 12.74, 12.72; HRMS calcd for C15H14N2O2S: 286.0771. Found
286.0774.
4.1.20. 1-Ethyl-5-(3-naphthalen-1-yl-allylidene)-2-thioxo-dihydro-pyrimidine-4,6-dione (8g)
Yield 81% (E & Z isomers); orange solid; mp 235 oC; 1H NMR (500 MHz, DMSO-d6) δ [ppm]:
8.82 (t, J = 15.0 Hz, 1H), 8.64 (dd, J = 15.0, 10.0 Hz, 1H), 8.52 (dd, J = 8.0, 2.5 Hz, 1H), 8.38
(d, J = 12.0 Hz, 1H), 8.14–8.02 (m, 3H), 7.70–7.60 (m, 3H), 4.40–4.30 (m, 2H), 1.20 (q, J = 7.0
Hz, 3H); 13C NMR (125 MHz, DMSO-d6) δ [ppm]: 179.29, 179.21, 161.49, 160.76, 160.35,
159.91, 156.83, 156.26, 151.09, 150.69, 134.00, 132.59, 132.52, 132.42, 131.71, 129.34, 127.93,
127.26, 127.04, 126.43, 126.37, 126.31, 123.79, 116.61, 116.41, 41.70, 41.35, 12.75; HRMS
calcd for C19H16N2O2S: 336.0927. Found 336.0932.
4.1.21.
1-Ethyl-5-(5-furan-2-yl-penta-2,4-dienylidene)-2-thioxo-dihydro-pyrimidine-4,6-dione
(8h)
21

ACCEPTED MANUSCRIPT
o
1
Yield 82% (E and Z isomers); reddish solid; mp 221 C; H NMR (500 MHz, DMSO-d6) δ
[ppm]: 12.38, 12.33 (2s, 2 × NH), 8.02–7.96 (m, 2H), 7.90–7.88 (m, 1H), 7.62–7.55 (m, 1H),
7.12–7.00 (m, 2H), 6.96–6.92 (m, 1H), 6.70–6.68 (m, 1H), 4.35–4.25 (m, 2H), 1.20–1.10 (m,
3H); 13C NMR (125 MHz, DMSO-d6) δ [ppm]: 179.00, 160.59, 160.37, 159.79, 156.15, 156.02,
155.60, 152.57, 146.63, 146.58, 130.75, 130.67, 129.44, 129.22, 126.88, 126.85, 115.71, 115.55,
114.80, 113.78, 41.62, 41.23, 12.76, 12.74; HRMS calcd for C15H14N2O3S: 302.0720. Found
302.0725.
4.1.22. 1,3-Diethyl-5-(3-furan-2-yl-allylidene)-2-thioxo-dihydro-pyrimidine-4,6-dione (8i)
1
Yield 78%; brown solid; mp 156oC; H NMR (500 MHz, CDCl3) δ [ppm]: 8.45 (dd, J = 15.5,
12.5 Hz, 1H), 8.16 (d, J = 12.5 Hz, 1H), 7.64 (d, J = 1.5 Hz, 1H), 7.22 (d, J = 15.0 Hz, 1H), 6.90
13
(d, J = 3.5 Hz, 1H), 6.60 (dd, J = 3.5, 2.0 Hz, 1H), 4.60–4.55 (m, 4H), 1.35–1.25 (m, 6H); C
NMR (125 MHz, CDCl3) δ [ppm]: 178.93, 160.61, 159.66, 157.57, 152.37, 147.08, 139.16,
123.96, 118.32, 114.79, 113.58, 43.72, 43.21, 12.45, 12.41; HRMS calcd for C15H16N2O3S:
304.0876. Found 304.0874.
4.1.23. 1,3-Diethyl-5-(3-phenyl-allylidene)-2-thioxo-dihydro-pyrimidine-4,6-dione (8j)
Yield 89%; reddish brown solid; mp 164 oC; 1H NMR (500 MHz, CDCl3) δ [ppm]: 8.62 (dd, J =
15.5, 12.0 Hz, 1H), 8.22 (d, J = 12.0 Hz, 1H), 7.72–7.66 (m, 2H), 7.46–7.40 (m, 4H), 4.60–4.55
13
(m, 4H), 1.35–1.25 (m, 6H); C NMR (125 MHz, CDCl3) δ [ppm]: 178.97, 160.52, 159.66,
158.48, 154.92, 135.37, 131.74, 129.30, 129.16, 125.68, 115.37, 43.77, 43.25, 12.45, 12.40;
HRMS calcd for C17H18N2O2S: 314.1084. Found 314.1085.
22

ACCEPTED MANUSCRIPT
4.1.24. 1-Allyl-3-ethyl-5-(3-furan-2-yl-allylidene)-2-thioxo-dihydro-pyrimidine-4,6-dione (8k)
1
Yield 92% (E and Z isomers); yellow solid; mp 143oC; H NMR (500 MHz, CDCl3) δ [ppm]:
8.52–8.44 (m, 1H), 8.20 (dd, J = 13.0, 3.5 Hz, 1H), 7.68 (t, J = 2.5 Hz, 1H), 7.24 (dd, J = 15.0,
3.5 Hz, 1H), 6.92 (d, J = 3.5 Hz, 1H), 6.10–6.00 (m, 1H), 6.02–5.94 (m, 1H), 5.36–5.30 (m, 1H),
13
5.28–5.25 (m, 1H), 5.18–5.14 (m, 2H), 4.62–4.56 (m, 2H), 1.36–1.32 (m, 3H); C NMR (125
MHz, CDCl3) δ [ppm]: 179.05, 179.00, 160.69, 160.57, 159.67, 159.61, 157.92, 157.89, 152.37,
147.16, 139.37, 139.36, 131.62, 131.49, 123.99, 123.93, 118.48, 118.25, 118.13, 114.59, 114.57,
113.61, 50.07, 49.56, 43.88, 43.37, 12.42, 12.37; HRMS calcd for C16H16N2O3S: 316.0876.
Found 316.0875.
4.1.25. 1-Allyl-3-ethyl-5-(3-phenyl-allylidene)-2-thioxo-dihydro-pyrimidine-4,6-dione (8l)
1
Yield 82% (E and Z isomers); reddish solid; mp 146oC; H NMR (500 MHz, CDCl3) δ [ppm]:
8.70–8.62 (m, 1H), 8.30–8.26 (m, 1H), 7.74–7.72 (m, 2H), 7.54–7.44 (m, 4H), 6.12–5.96 (m,
13
1H), 5.36–5.26 (m, 2H), 5.20–5.16 (m, 2H), 4.62–4.56 (m, 2H), 1.38–1.32 (m, 3H); C NMR
(125 MHz, CDCl3) δ [ppm]: 179.07, 179.02, 160.60, 160.48, 159.67, 159.61, 158.88, 158.84,
155.24, 155.22, 135.32, 131.82, 131.53, 131.41, 129.37, 129.35, 129.18, 129.17, 125.70, 125.64,
118.35, 118.16, 115.16, 115.13, 50.11, 49.61, 43.94, 43.42, 12.42, 12.37; HRMS calcd for
C18H18N2O2S: 326.1084. Found 326.1086.
4.1.26.
1-Allyl-3-ethyl-5-(3-thiophen-2-yl-allylidene)-2-thioxo-dihydro-pyrimidine-4,6-dione
(8m)
1
Yield 90% (E and Z isomers); yellow solid; mp 146oC; H NMR (500 MHz, CDCl3) δ [ppm]:
8.46–8.38 (m, 1H), 8.20 (dd, J = 12.5, 4.0 Hz, 1H), 7.68–7.58 (m, 2H), 7.54–7.38 (m, 1H),
23

ACCEPTED MANUSCRIPT
7.18–7.12 (m, 1H), 6.02–5.92 (m, 1H), 5.34–5.24 (m, 2H), 5.18–5.14 (m, 2H), 4.60–4.54 (m,
2H), 1.36–1.30 (m, 3H); 13C NMR (125 MHz, CDCl3) δ [ppm]: 179.03, 178.99, 160.66, 160.54,
159.73, 159.66, 158.36, 158.31, 146.98, 144.26, 141.35, 141.33, 132.36, 132.34, 131.61, 131.48,
128.96, 128.51, 125.08, 125.02, 118.25, 118.00, 50.08, 49.53, 43.90, 43.34, 12.44, 12.37; HRMS
calcd for C16H16N2O2S2: 332.0648. Found 332.0652.
4.1.27. 1,3-Diallyl-5-(3-furan-2-yl-allylidene)-2-thioxo-dihydro-pyrimidine-4,6-dione (8n)
Yield 89%; reddish brown solid; mp 135oC; 1H NMR (500 MHz, CDCl3) δ [ppm]: 8.45 (dd, J =
15.0, 12.5 Hz, 1H), 8.18 (d, J = 12.5 Hz, 1H), 7.64 (d, J = 1.5 Hz, 1H), 7.22 (d, J = 15.0 Hz,
1H), 6.92 (d, J = 3.5 Hz, 1H), 6.60 (dd, J = 3.5, 1.5 Hz, 1H), 6.00–5.90 (m, 2H), 5.34–5.24 (m,
13
4H), 5.18–5.14 (m, 4H); C NMR (125 MHz, CDCl3) δ [ppm]: 179.10, 160.66, 159.63, 158.28,
152.35, 147.28, 139.63, 131.50, 131.38, 123.95, 118.70, 118.29, 118.17, 114.33, 113.67, 50.22,
49.70; HRMS calcd for C15H16N2O3S: 328.0876. Found 328.0878.
4.1.28. 1,3-Diallyl-5-(3-phenyl-allylidene)-2-thioxo-dihydro-pyrimidine-4,6-dione (8o)
o
1
Yield 87%; yellow solid; mp 154 C; H NMR (500 MHz, CDCl3) δ [ppm]: 8.55 (dd, J = 15.5,
12.0 Hz, 1H), 8.28 (d, J = 12.0 Hz, 1H), 7.72 (dd, J = 7.0, 1.0 Hz, 2H), 7.52–7.40 (m, 4H), 6.00–
13
5.90 (m, 2H), 5.35–5.25 (m, 4H), 5.20–5.15 (m, 4H); C NMR (125 MHz, CDCl3) δ [ppm]:
179.13, 160.57, 159.62, 159.23, 155.52, 135.29, 131.90, 131.42, 131.30, 129.42, 129.18, 125.67,
118.38, 118.21, 114.94, 50.25, 49.74; HRMS calcd for C19H18N2O2S: 338.1084. Found
338.1088.
4.2. Biological evaluations
24

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4.2.1. Reagents and antibodies
Antibodies for Western blot purposes were purchased from cell signaling [anti-PARP (9542s), p-
Bcl-2 (2827s), survivin (2802s) and Bcl-xL (2764s)] and Sigma-Aldrich [β-actin (A5316)]. MTT
(Thiazolyl Blue Tetrazolium Bromide) was obtained from Sigma-Aldrich (M5655−500MG).
4.2.2. Cell culture conditions
WM2664, CHl-1, A375M, MDA-MB-231, PANC-1, MCF-7, 1205Lu cell lines were maintained
in DMEM medium, while HCT116 cells were maintained in McCoy’s 5A medium. Both the
mediums were supplemented with 10% fetal bovine serum (FBS) and 100 units/mL of penicillin
and streptomycin at 37 °C. MCF10A cells were maintained in MEGM™ Mammary Epithelial
Cell Growth Medium (Lonza, CC-3151 & CC-4136) at 37 °C.
4.2.3. Cell viability assay
The effects of thio- and seleno-barbituric acid derivatives (2, 5, and 8) on the cell viability were
determined by the MTT uptake method as described before.[29] Briefly, 3,000 cells were
incubated with various concentrations of thiobarbiturate derivatives in triplicate in a 96-well plate
for 48 h at 37 °C. MTT solution (5 mg/mL in PBS) was added to each well and incubated for 3 h at
37 °C. After 3 h, DMSO was added to dissolve the resultant formazan crystal and the optical
density was measured at 570 nm using a 96-well multiscanner (Dynex Technologies, MRX
Revelation; Chantilly, VA, USA). Backgrounds were subtracted at 630 nm. IC50 value for each
compound was determined by at least three independent experiments and represented with a
standard error (Table 1).
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4.2.4. Live/dead assay
To measure apoptosis, we used the Live/Dead assay as described before.[29] Live/Dead assay
determines intracellular esterase activity and plasma membrane integrity. Calcein-AM, a
nonfluorescent polyanionic dye, is retained by live cells, in which it produces intense green
fluorescence through enzymatic (esterase) conversion. In addition, the ethidium homodimer
enters cells with damaged membranes and binds to nucleic acids, thereby producing a bright red
fluorescence in dead cells. Briefly, 2 x 105 cells were incubated with mentioned amounts of 2b
and 8b for 24 h at 37 °C. Cells were stained with the Live/Dead reagent (5 µM ethidium
homodimer and 5 µM calcein-AM) and incubated at 37 °C for 30 min following manufacture's
protocol as described earlier. Cells were analyzed under a fluorescence microscope (Labophot-2;
Nikon) as described before.6
4.2.5. Western blot analysis
To determine the effect of 2b and 8b on apoptotic proteins, whole cell lysates were prepared as
previously described.[29] The whole cell lysates were resolved by SDS-PAGE. After
electrophoresis, the proteins were electro-transferred to PVDF membranes, blotted with the
relevant antibodies, and detected by an enhanced chemiluminescence reagent.
4.2.6. Annexin V assay
To determine the ability of 2b and 8b on apoptosis, UACC903 and CHL-1 (5 × 105) cells were
treated with 25 and 50 µM with 2b and 8b for 24h. Muse Annexin V & Dead Cell kit (Millipore,
Catlog No, MCH100105) was used to detect live, early apoptotic, late apoptotic and necrotic
cells in both floating and adherent cells as previously described,[29] using Muse cell analyzer
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(EMD Millipore, Billerica, MA, USA). Muse Annexin V kit contains 7-AAD, which enters any
cells that have lost their plasma membrane integrity, and hence marks dead cells. The kit also
contains Annexin V dye, which stains phosphatidylserine (PS) on the outer surface of apoptotic
cells. Data acquired from the equipment was analyzed using Muse 1.4 software.
4.2.7. Caspase 3/7 activity assay
To establish whether 2b and 8b induce caspase 3/7 activity, UACC903 and CHL-1 (5 × 105) cells
were treated with 25 and 50 µM with 2b and 8b for 24 h. Caspase 3/7 activity was monitored
using Muse Caspase-3/7 Assay kit with Muse cell analyzer (EMD Millipore, Billercia, MA,
USA) as previously described.[29] Muse Caspase 3/7 kit contains a cell permeable reagent,
NucView, that can detect active caspase 3/7. This reagent has a DNA binding dye that is
connected to a DEVD peptide substrate, which inhibits the DNA dye from binding to the DNA.
However, in presence of active caspase 3/7, DEVD peptide gets cleaved and the DNA binding
dye is released and fluorescence when bound with DNA. The kit also contains 7-AAD, a dead
cell marker. Muse 1.4 software was used to analyze the data.
Acknowledgement
The authors thank the Department of Pharmacology, Penn State College of Medicine, and Penn
State Cancer Institute for financial support. This study was also supported partially by NIH’s
NCI Grant R21 CA167406-02 & Elsa U. Pardee Foundation Grant (SYL). The authors thank Dr.
Jyh-Ming Lin, Solution Phase NMR Facility at Core Research Facilities of the Penn State
College of Medicine for recording NMR spectra.
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Supplementary material. Copies of the 1H NMR and 13C NMR spectra for new compounds 2a-
f, 5a-c, and 8a-o.
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