New thiobarbituric acid scaffold-based small molecules: Synthesis, cytotoxicity, 2D-QSAR, pharmacophore modelling and in-silico ADME screening

Article abstract of DOI:10.1016/j.ejps.2019.01.023

A series of twenty five new thiobarbituric acid derivatives, viz. 3a-h, 4–7, 8a-c, 9, 10a-c, 11 and 12a-d, were designed and synthesized as potential cytotoxic agents. In-vitro screening of the new compounds against the three human cancer cell lines Caco-

Full text of DOI:10.1016/j.ejps.2019.01.023

Accepted Manuscript
New thiobarbituric acid scaffold-based small molecules:
Synthesis, cytotoxicity, 2D-QSAR, pharmacophore modelling and
in-silico ADME screening
Heba S.A. El-Zahabi, Maha M.A. Khalifa, Yomna M.H. Gado,
Amel M. Farrag, Mahmoud M. Elaasser, Nesreen A. Safwat,
Reham R. AbdelRaouf, Reem K. Arafa
PII:
DOI:
Reference:
S0928-0987(19)30027-2
https://doi.org/10.1016/j.ejps.2019.01.023
PHASCI 4814
To appear in:
European Journal of Pharmaceutical Sciences
Received date:
Revised date:
Accepted date:
7 December 2018
12 January 2019
18 January 2019
Please cite this article as: H.S.A. El-Zahabi, M.M.A. Khalifa, Y.M.H. Gado, et al., New
thiobarbituric acid scaffold-based small molecules: Synthesis, cytotoxicity, 2D-QSAR,
pharmacophore modelling and in-silico ADME screening, European Journal of
Pharmaceutical Sciences, https://doi.org/10.1016/j.ejps.2019.01.023
This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.

ACCEPTED MANUSCRIPT
New Thiobarbituric Acid Scaffold-based Small Molecules: Synthesis,
Cytotoxicity, 2D-QSAR, Pharmacophore Modeling and in-silico
ADME Screening
Heba S.A. El-Zahabia, Maha M. A. Khalifaa, Yomna M. H. Gadoa, Amel M. Farraga,
Mahmoud M. Elaasserb, Nesreen A. Safwatb, Reham R. AbdelRaouf,c Reem K. Arafac,*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University, Cairo 11884, Egypt.
b The Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt.
c Biomedical Sciences Program, Zewail City of Science and Technology, 12578, Cairo, Egypt.
Abstract
A series of twenty five new thiobarbituric acid derivatives, viz. 3a-h, 4-7, 8a-c, 9, 10a-c, 11 and
12a-d, were designed and synthesized as potential cytotoxic agents. In-vitro screening of the new
compounds against the three human cancer cell lines Caco-2, HepG-2 and MCF-7 was performed to
assess their intrinsic activity. Compound 12d exhibited potent sub-micromolar activity against HepG-2
and MCF-7 (IC50 = 0.07 and 0.08 µM, respectively). In-silico pharmacophore modelling of this
chemotype compounds disclosed a five features’ pharmacophore model representing essential steric
and electronic fingerprints essential for activity. Finally, a 2D-QSAR model was devised to
quantitatively correlate the 2D molecular feature descriptors of this series of thiobarbiturates with their
cytotoxic activity against MCF-7. Finally, in silico evaluation of the physicochemical and ADME
properties of these derivatives was performed.
Key words: Thiobarbituric acid derivatives; Merbarone; cytotoxic activity; pharmacophore; 2D-
QSAR.
*Corresponding author
Reem K. Arafa
Professor of Biomedical Sciences
University of Science and Technology
Zewail City of Science and Technology
Ahmed Zewail Road
October Gardens, 6th of October City, Giza, Egypt, 12578
Mobile: +2-01002074028
E-mail: rkhidr@zewailcity.edu.eg
1

ACCEPTED MANUSCRIPT
1. Introduction
Thiobarbiturate scaffold based small molecules have been reported to possess antitumor
capacity with the observed activity being attributed to targeting different enzymes having an important
role in cancer development[1-11] e.g. topoisomerase II[1], sirtuins[6-8, 10] and B-Raf protein kinase[9].
National Cancer Institute (NCI/USA) reported the thiobarbiturate analogue Merbarone I as
potent antitumor agent in fighting melanoma and sarcoma cancer via inhibiting topoisomerase II[1, 4].
In addition, other thiobarbiturate derivatives e.g. II[6], III[10] and IV[10] displayed remarkable activities
against cancer cell lines via inhibiting Sirt1 and/or Sirt2 enzymes (Figure 1). Focusing on B-Raf
kinase as a key enzyme that demonstrated mutations in a broad range of human cancers, B-RafV600E is
considered an important target for anticancer drugs. Recently, novel thiobarbiturate derivatives V and
VI were reported as B-RafV600E inhibitors (Figure 1)[9].
In medicinal chemistry, the thiobarbiturate ring represents an excellent scaffold being
[1-14],
[15-17]
incorporated in many biologically active small molecules, e.g. antitumor agents
antivirals
and antimicrobials [18-21], in addition to the well-known clinically used CNS depressant
thio/barbiturates. Structurally speaking, compounds II-VI chemotype can be envisaged as having two
subdomains, a non-modified chemical moiety (domain A, thiobarbituric scaffold) and a C5 modifiable
domain B with a methylidene bridge as a linker. Domain B is represented by aryl or heteroaryl groups
(Figure 1).
Modification of moiety B was attributed to the replacement of the furfuryl nucleus in V and VI
with aliphatic, aryl and heteroaryl groups. Compounds 4- 6 and 9 included the replacement of furfuryl
nucleus (moiety B) in V and VI with thiobarbituric acid motif using either aliphatic or aryl diamine
linkers. Modification in compounds 2, 3 a-h, 7, 10 a-c, 11 and 12a-d, represented replacement of B by
(aryl/heteroaryl) groups. Simultaneously, various amines (amino group, hydrazone,
hydrazinocarbonyl, hydrazinosulfone and thiosemicarbazide moieties) were applied as a linker. The
last modification, displayed isosteric switching of furfuryl ring in V and VI to pyrazole ring in 8a-c.
(Figure 2).
Based on these findings and in line with our continuous investigation for anticancer agents
with variable chemical scaffolds [19-21], a new series of C5 substituted thiobarbiturates was designed
and synthesized with a focus on maintaining the thiobarbituric scaffold and exchanging methylidene-
bridge attached substituents at C5 of the thiobarbiturate ring. Biological assessment of the cytotoxic
ability of these new compounds was performed against 3 human cancer cell lines, viz. Caco-2
(colorectal cancer), HepG-2 (liver cancer) and MCF-7 (breast cancer). Further, pharmacophore
modelling was carried out to extract the important steric and electronic features of this class of
compounds underlying their inherent pharmacological activity. Subsequently, a 2D-QSAR model
based on the MCF-7 cytotoxicity data was built to quantitatively correlate 2D structural features
descriptors with the observed cytotoxicity. Finally, in silico assessment of the physicochemical and
ADME properties of all compounds was performed.
2

ACCEPTED MANUSCRIPT
Figure 1: Merbarone I and thiobarbiturate analogues with promising anticancer activity
2. Result and discussion
2.1. Chemistry
Synthesis of the target compounds is depicted in schemes 1-5. 5-
[(Dimethylamino)methylidene]-2-thioxodihydropyrimidine-4,6(1H,3H)-dione (2) was prepared by
reacting thiobarbituric acid 1 with N,Nʹ-dimethylformamide dimethylacetal (DMF-DMA) in dry
[22,23]
1
benezene, adopting the reported condition
(Scheme 1). H-NMR spectrum of 2 revealed the
presence of two signals at δ 3.50 and 7.88 ppm for the protons corresponding to the di-CH3 groups and
vinylic (=CH-N) proton, respectively. The mass spectrum showed a molecular ion peak [M+] at m/z:
199. The IR chart displayed bands at 3102 and 1603 cm-1 representing to the NH and CO groups,
respectively.
Precursor 2 was reacted with different aromatic amines adopting the reported procedure[24] to
afford the corresponding dihydro-5-[(arylamino)methylidene]-2-thioxopyrimidine-4,6(1H,3H)-dione
3a-h (Scheme 1). The proposed structures of 3a-h were compatible with their elemental and spectral
data where their 1H-NMR spectra showed the disappearance of the singlet signal at δ 3.50 ppm for the
protons of the di-CH3 groups, in each case. Mass spectra of 3a-h illustrated a peak for each expected
molecular ion (Experimental section).
3

ACCEPTED MANUSCRIPT
Scheme 1: Synthesis of derivatives 3a-h.
In a similar manner, precursor 2 was reacted with different diamines, viz. piperazine, 1,4-
phenylendiamine and ethylenediamine, to afford bis[2-thioxo-dihydropyrimidine-4,6(1H,3H)-dione]
derivatives 4-6 (Scheme 2). 1H-NMR spectrum of N,N-bis[5-methylidene-2-thioxo-
dihydropyrimidine-4,6(1H,3H)-dione]piperazine 4 revealed a multiplet signal at δ 3.07-3.09 ppm
1
characterizing aliphatic protons of the piperazine ring. Concomitantly, H-NMR spectrum of 5
revealed two doublets at δ 6.61 ppm and 7.16 ppm (J= 8.7 Hz) assigned to the aromatic ring protons
and a signal appeared at δ 8.38 ppm for the vinylic proton (=CH-N).
Scheme 2: Synthesis of derivatives 4-6.
Upon reacting compound 2 with one mole equivalent ratio of hydrazine hydrate in refluxing
ethanol,
5-(hydrazinylmethylidene)-2-thioxo-dihydropyrimidine-4,6(1H,3H)-dione
7
was
afforded[25,26]. Subsequently, reaction of 7 with diethylmalonate, ethyl acetoacetate or ethyl
cyanoacetate was carried in acetic acid under reflux to afford the corresponding cycloaddition
pyrazolyl products 8a-c, respectively (Scheme 3). 1H-NMR spectrum of 8a revealed the appearance of
4

ACCEPTED MANUSCRIPT
singlet at δ 2.89 ppm, integrated for two protons at C4 of the pyrazole ring. Mass spectrum illustrated
a molecular ion peak [M+] at m/z 254. 1H-NMR spectra of 8b and 8c exhibited a singlet at δ 5.63 ppm
1
and 7.47 ppm corresponding for the vinylic proton at C4 of the pyrazole ring, respectively. Also, H-
NMR of 8b revealed a signal at δ 2.22 ppm for the CH3 group at C5 of the pyrazole ring, while 8c
expressed D2O exchangeable singlet at δ 11.31 ppm for NH2 protons. 13C-NMR for 8b showed a
signal at 145.18 ppm representing the vinylic carbon and three signals at 158.00, 161.90 and 163.00
ppm representing three carbonyl carbons. Mass spectra of 8b and 8c showed molecular ion peaks
[M++1] at m/z: 253 and 254, respectively.
Scheme 3: Synthesis of derivatives 7 and 8a-c.
5

ACCEPTED MANUSCRIPT
Reacting equimolar ratio of 2 and hydrazinylmethylidene pyrimidine 7 in refluxing ethanol
afforded
N,N-bis(5-methylidene-2-thioxo-dihydropyrimidine-4,6(1H,3H)-dione)hydrazine
9.
Noticeably, compound 9 was not successfully obtained upon treating the precursor 2 with hydrazine
hydrate in 2:1 mole equivalence (Scheme 4). Elemental and spectral data supported the assigned
structure of bis-pyrimidine hydrazine derivative 9.
Scheme 4: Synthesis of derivative 9.
Scheme 5 depicts the synthesis of derivatives 10a-c, 11 and 12a-d. Reaction of
hydrazinylmethylidenepyrimidine 7 with bromobenzenesulfonyl chloride or benzoyl chlorides
adopting the reported method[27] furnished the corresponding benzenesulfonohydrazide and
benzohydrazides 10a-c, respectively. Spectral and elemental analyses were compatible with the
1
structures of 10a-c, for example, H-NMR spectrum of 10c showed two doublets at δ 7.60 and 7.92
ppm (J= 7.2 Hz) corresponding to the aromatic protons. Furthermore, compound 7 was reacted with
phenyl isothiocyanate to afford the corresponding phenylthiosemicarbazide 11. Spectral and elemental
1
analyses ascertained the structure of 11. H-NMR spectrum of 11 showed three multiplets integrated
for five aromatic protons at δ 7.05-7.10, 7.32-7.39 and 7.58-7.65 ppm. Mass spectrum showed the
molecular ion peak [M++1] at m/z: 322.
Finally, condensation of 7 with different aromatic aldehydes in refluxing ethanol afforded
benzylidenehydrazinylmethylidene derivatives 12a-d.[24,28-31] Compounds 12a-d gave satisfactory
1
analytical and spectral data in accordance with their depicted structures. H-NMR of 12a showed two
1
signals at 7.46 ppm and 8.29 ppm assigned for the two vinylic protons. H-NMR of 12b showed the
methoxy protons signal at 3.74 ppm while in 12c the two methoxy protons appeared as two singlets at
3.80 and 3.82 ppm. 13C-NMR of 12c showed the two vinylic carbons at 155.56 and 159.35 ppm while
the carbonyl carbon appeared at 161.62 ppm.
6

ACCEPTED MANUSCRIPT
Scheme 5: Synthesis of derivatives 10a-c, 11 and 12a-d.
2.2. Pharmacology
2.2.1 Antitumor activity
Anticancer activity of the target compounds 2, 3a-h, 4-7, 8a-c, 9, 10a-c, 11 and 12a-d was
evaluated against human colorectal cancer (Caco-2), hepatic cancer (HepG-2) and breast cancer
(MCF-7) which were synthesized on the light of the promising anticancer activity of thiobarbiturates
V and VI [9]. Doxorubicin was used as a reference standard and showed IC50 of 34.9, 4.68 and 1.17 µM
against the tested cancer cell lines, respectively. A comparative structural profile between (V, VI) and
the target compounds 2, 3a-h, 4, 5, 6, 7, 8a-c, 9, 10a-c, 11 and 12a-d was illustrated in Figure 2.
Structural features of V and VI included non-modified moiety A (thiobarbituric acid motif) and
modified moiety B (colored red and blue) with a linker of methylidene group at C5 of thiobarbituric
acid (Figure 2).
All the test compounds except precursor 2 showed potent anticancer activity against the
screened cancer cell lines (Table 1). With regards to cytotoxicity against Caco-2 cell line, the
anticancer effect of the target compounds 3a-h, 7, 10a-c, 11 and 12a-d surpassed that of doxorubicin.
Among the test compounds, benzenesulfonohydrazide 10a was the most potent compound (IC50 = 0.11
µM) compared to doxorubicin (IC50 = 34.9 µM). On the other hand, the aminopyrazolone derivative 8c
showed the lowest IC50 (1.14 = µM), in spite of being still 34 times more active than doxorubicin.
Also, the benzohydrazide 10b and the benzylidenehydrazinyl derivative 12a showed equipotent
activity against Caco-2 cell line (IC50 = 0.16 µM each).
Similarly, Hepatic cell line HepG-2 showed high sensitivity towards the test compounds
compared to doxorubicin. Comparatively, thiophenylmethylidene derivative 12d was several folds
more active than doxorubicin (IC50 of 0.07 and 4.68 µM, respectively), while
phenylthiosemicarbazide, 11 showed the lowest activity of IC50 = 0.89 µM. Further, compounds 3a,b,
10a,b and 12a explored almost half the activity of 12d (IC50 = 0.18, 0.16, 0.16, 0.14 and 0.18 µM,
respectively) compared to doxorubicin. Thus these compounds recorded forty folds the activity of
doxorubicin (IC50 of 4.68 µM).
7

ACCEPTED MANUSCRIPT
Simultaneously, all the test compounds except 2 showed potent anticancer activity against
(MCF-7) compared to doxorubicin (IC50
1.17 µM). Among the test compounds,
0.08 µM) was several folds more potent than
=
thiophenylmethylidene derivative 12d (IC50
=
doxorubicin, while bis-thiobarbituricpiperazine derivative 4 showed the lowest activity of IC50 = 0.81
µM compared to doxorubicin. Also compounds 3a, 3e, 5 and 10c almost demonstrated half the activity
of 12d (IC50 = 0.15, 0.18, 0.17 and 0.12 µM, respectively), yet they were almost ten folds more active
than doxorubicin.
Table 1: IC50 of the test set of compounds against human colorectal cancer (Caco-2), hepatic cancer (HepG-2),
and breast cancer (MCF-7).
IC50 (µM)
Compd #
Caco-2
HepG-2
MCF-7
N.A.a
0.57b
0.24
0.64
0.32
0.26
0.36
0.39
0.24
0.91
0.23
0.51
0.74
0.18
0.38
1.14
0.51
0.11
0.16
0.52
0.4
N.A.
0.18
0.16
0.71
0.29
0.28
0.52
0.46
0.29
0.79
0.21
0.52
0.52
0.25
0.63
0.66
0.37
0.16
0.14
0.40
0.89
0.18
0.49
0.56
0.07
4.68
N.A.
0.15
0.28
0.53
0.26
0.18
0.45
0.36
0.24
0.81
0.17
0.42
0.35
0.57
0.33
0.36
0.27
0.23
0.27
0.12
0.58
0.32
0.35
0.41
0.08
1.17
2
3a
3b
3c
3d
3e
3f
3g
3h
4
5
6
7
8a
8b
8c
9
10a
10b
10c
11
12a
12b
12c
12d
0.16
0.36
0.70
0.46
34.9
Doxorubicin
a N.A. = Not active
b IC50 values are the average of 3 independent runs.
2.3. Structural Design and Structure Activity Relationship (SAR)
Compounds V and VI were adopted as structural leads for design of new compounds in this
study (figure 2). Molecular manipulations performed aimed to examine the effect of replacement of
the arylfuranyl moiety by classical and non-classical bioisosteres. Also attempted was the synthesis of
bis-compounds with double thiobarbiturate warheads. Replacement of the heteroaryl moiety B of V
and VI with dimethylamino moiety in precursor 2, completely abolished the anticancer activity
8

ACCEPTED MANUSCRIPT
reflecting the possible need for a hydrogen bond donor and/or an aromatic side chain linked to the
thiobarbiturate ring for activity. Subsequently, 2 was derivatized to furnish the compounds in this
study 3a-h, 4-7, 8a-c, 9, 10a-c, 11 and 12a-d. All adopted synthetic modifications retrieved the
cytotoxic activity of these series. Structural modification profiling of modified moiety B of the test
compounds demonstrates variable hydrophilic/hydrophobic and hydrogen bond donor/acceptor
characters.
First, derivatives 3a-h had their aryl/heteroaryl terminal rings attached through an amino
group to the thiobarbiturate warhead in place of the furan ring in V and VI. The hydrazinyl analog 7
and its arylidene derivatives 12a-c had an aliphatic/hydrogen bond donor linker. A highlight was the
thienylmethylidene derivative 12d that was several times more potent than doxorubicin against Caco-2
(IC50 = 0.46 µM), HepG-2 (IC50 = 0.07 µM) and MCF-7 (IC50 = 0.08 µM) and thus the most potent
compound in this study. Replacement of the terminal methylidene group by a hydrogen bond acceptor
moiety in 10a-c, C=O or SO2, lead to enhanced potency. Linker length extension in 11 was much in
favor of cytotoxicity. (Figure 2, upper panel)
Elimination of the terminal aryl ring and replacement of the furan ring by a pyrazoline or
pyrazolidine ring decorated with hydrogen bond donor/acceptor groups furnished derivatives 8a-c with
retained but moderate to low efficacy. (Figure 2, middle panel)
Finally, synthesis of derivatives with double thiobarbiturate warheads and with variable
aromatic/cyclic or acyclic aliphatic diamine linkers furnished compounds 4-6 and 9. While this
strategy didn’t display specific enhancement in activity, the most potent derivative was that with the
aromatic phenylenediamine linker (compound 5). Compound 9, with the hydrazinyl linker, was less
potent than 5, but the least potent in this series were derivatives 4 and 6 having alicyclic or aliphatic
diamine linker groups, respectively. (Figure 2, lower panel)
9

ACCEPTED MANUSCRIPT
10

ACCEPTED MANUSCRIPT
Figure 2: Strucutral design of the target thiobarbiturate derivatives. Fragment replacement profile of reported
thiobarbiturates (V, VI) and designed thiobarbituric acid derivatives as antitumor. First modification (upper
panel) in compounds 2, 3a-h, 7, 10a-c, 11 and 12a-d, moiety B was replaced by different amines e.g. amino
group, hydrazone, hydrazinocarbonyl, hydrazinosulfone and thiosemicarbazide moieties. Second modification
in compounds 8a-c (middle panel) explored isosteric replacement of furan ring by pyrazole moiety.
Thirdmodification (lower panel) for compounds 4-6 and 9 included replacing moiety B in V and VI with
aliphatic/aryl diamine linker to obtain bis-thiobarbiturates.
2.4. Quantitative Structure Activity Relationship (QSAR)
A group of eighteen compounds with a variety in structure and IC50 values was used as a
training set to build a pharmacophore model based on their 2D descriptors. The built in set of 180 2D-
descriptors in MOE (version 2016.08)[32] was calculated for all the compounds. Weka (version
3.8.3)[33-34] and MOE analysis were used to filter the most important 2D-descriptors. Those selected as
most influential were utilized for building various pharmacophore models by MOE by making
alternatives of all sets and further filtration by exclusion of the least important descriptors.
The best predicting model consisted of four 2D descriptors of two different types; adjacency
and distance matrix descriptors (BCUT_SMR_0) and partial charge descriptors (PEOE_VSA-2,
PEOE_VSA+3 and PEOE_VSA_FNEG) (more details about the descriptors are provided in the
supplementary material section). Statistically, the best model showed correlation coefficient of 75.55%
(r2= 0.7555), an internal predictive power by cross validation of 64% with XRMSE= 0.1818,
(r2=0.6402) (Table 2 and figure 3). It also displayed a predictive power of the external test set (6
compounds) of 70.44% (r2= 0.7044) (Table 3 and figure 4).
The estimated linear regression equation of the model is:
pIC50 = 8.01089 + (1.42041 * BCUT_SMR_0) + (2.68121 * PEOE_VSA_FNEG) + (0.02087 *
PEOE_VSA+3) + (0.01037 * PEOE_VSA-2)
The equation shows that all the four descriptors correlate positively with pIC50.
11

ACCEPTED MANUSCRIPT
Table 2: Compounds in the training set with their pIC50 values (MCF-7 cell line), predicted pIC50, residuals and
Z-scores.
PRED
pIC50
6.54
6.66
6.45
6.51
6.42
6.34
6.63
6.24
6.15
6.41
6.36
6.39
6.79
6.94
6.34
6.77
6.36
7.16
XZ-
SCORE*
1.68
1.21
2.72
0.21
1.05
0.02
0.46
2.23
1.38
1.61
1.23
0.38
0.08
1.09
2.63
0.25
0.86
0.18
Compd #
pIC50
RES
Z-SCORE
XPRED*
XRES*
6.74
6.80
6.15
6.54
6.55
6.34
6.68
6.28
6.28
6.60
6.20
6.43
6.80
6.85
6.05
6.74
6.25
7.15
0.20
0.14
-0.30
0.03
0.13
0.00
0.05
0.04
0.14
0.20
-0.16
0.04
0.01
-0.08
-0.29
-0.03
-0.11
0.00
1.42
0.98
2.13
0.19
0.94
0.02
0.36
0.29
0.96
1.38
1.10
0.29
0.04
0.57
2.06
0.20
0.76
0.02
6.52
6.62
6.49
6.51
6.40
6.34
6.61
5.96
6.09
6.38
6.37
6.38
6.78
7.01
6.38
6.78
6.38
7.18
0.23
0.17
-0.34
0.03
0.15
0.00
0.07
0.32
0.19
0.22
-0.17
0.05
0.01
-0.16
-0.33
-0.04
-0.12
-0.03
3a
3b
3c
3d
3e
3g
5
6
7
8a
8b
9
10a
10b
11
12a
12c
12d
* X: cross validation
Figure 3: Correlation plot of the training set showing R2 value = 0.755
Table 3: Compounds in the test set with their PIC50 values (MCF-7 cell line), predicted pIC50 and the
residual values.
Compd #
pIC50
6.28
6.54
6.10
6.18
6.40
PRED pIC50
6.29
RES
0.01
0.20
-0.19
0.09
0.63
3f
3h
4
8c
10c
6.74
5.92
6.27
7.03
12

ACCEPTED MANUSCRIPT
6.31
6.57
0.26
12b
Figure 4: Correlation plot of the test set showing R2 value= 0.704
2.5. Pharmacophore
For building the pharmacophore model, 15 compounds of thiobarbiturates were energy
minimized using the MMFFX94 forcefield and a gradient of 0.01 RMSD in MOE (version
2016.08). Alignment of the energy minimized molecules was then performed followed by
pharmacophore model building.
The model was built based on five main common structural features; PiR | Aro | Hyd, Acc,
Acc, Hyd & Don. For the internal validation, the model was able to identify all the compounds in
the test set except for the doxorubicin, which was expected because it represents a different
chemotype (Figures 5&6).
[35]
For external validation, OTAVA RNA binding database
was used as a decoy database
to test the model accuracy. It picked up only 127 out of 2775 as actives (a low percentage of
4.5%). This step assures the model’s selectivity.
13

ACCEPTED MANUSCRIPT
Figure 5: Five features of the pharmacophore model on the 15 aligned compounds.
Figure 6: 3D spatial distribution of the five pharmacophoric features.
2.6. In silico physicochemical and ADME properties prediction
The results of drug likeness and ADME prediction using SwissADME[36] are summarized
in table 4 and table 5, respectively.
With regards to drug likeness (table 4), most of the compounds have zero violation for
Lipinski’s rule for oral drugs, except for three compounds (5, 6 & 9) which have only one
violation (HBD >5) yet with which they still lie in the category of orally bioavailable compounds.
For Doxorubicin, it displayed 3 violations (HBD >5, HBA >10 and MWt >500). Most of TPSA
values were less than 200 Å2, except for the three compounds (having HBD violations) and
Doxorubicin which were found to be slightly higher than 200 Å2. Rotatable bonds are any single
bonds not in a ring, attached to a heavy atom (non-hydrogen). All compounds have between 1 to 5
rotatable bonds which is in favor of binding to their biotarget to avoid entropic penalty.
Table 4: Drug-likeness based on Lipinski’s rule of five, TPSA and number of rotatable bonds
# of
violations*
# of rot.
bonds
Molecule
HBD
HBA
MlogP
TPSA
14

ACCEPTED MANUSCRIPT
3
3
4
4
4
3
3
3
4
6
6
4
3
3
4
6
4
4
4
5
3
3
3
3
6
2
2
3
4
4
4
3
4
4
4
4
3
4
3
3
4
5
3
3
2
3
4
5
3
12
-0.36
0.6
-0.9
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
1
0
0
0
0
0
0
0
0
3
102.32
102.32
122.55
139.62
139.62
128.62
115.21
139.7
187.06
204.64
204.64
128.34
139.7
2
2
2
3
4
5
2
2
2
4
5
1
1
1
1
3
4
4
4
5
3
4
5
3
5
3a
3b
3c
3d
3e
3f
3g
3h
4
5
6
7
8a
8b
8c
9
10a
10b
10c
11
12a
12b
12c
12d
-1.79
-0.68
-0.15
-1.05
-0.46
-2.62
-1.8
-3.15
-2.35
-2.23
-1.25
-1.68
-2.91
-0.19
-0.55
0.4
128.08
154.1
204.64
156.87
131.42
131.42
158.47
114.68
123.91
133.14
142.92
206.07
-0.55
-0.42
-0.68
-0.94
-0.55
-2.1
Doxorubicin
*Violations considering MlogP, HBD, HBA and compound’s MWt (<500)
With regards to the pharmacokinetic properties and medicinal chemistry parameters of the
new compounds (table 5), it was found that there are nine compounds (3a-c, 3f, 3g, 10c and 12a-
c) having high gastrointestinal absorption compared to low absorption of doxorubicin. Also, all
compounds have no permeation to the blood brain barrier, thus ensuring that these systemically
targeted molecules will have low to no CNS side effects. The rate of GIT absorption and
permeability of BBB was predicted according to Boiled-Egg theory, where the yolk represents
BBB permeable compounds and the white represents GI absorption.
All the compounds, according to a SVM model with accuracy of 0.72 in internal validation
and 0.88 in external validation, were found to be poor substrates for P-glycoprotein (P-gp) except
for the Doxorubicin. Hence, the bioavailability scores were found to be higher in all compounds
than Doxorubicin.
In Pan-interference compounds assay (PAINS) structural alerts, all the compounds along
with Doxorubicin were found to have only one structural alert (quinone A for doxorubicin and
ene_six_het_A for other compounds). Though PAINS are important features to be considered
while developing drugs to avoid false positives results, yet over estimation and blind use of these
filters might only lead to exclusion of promising hits based on phantom PAINS.[37]
Synthetic accessibility score shows that thiobarbiturates in this study are more
synthetically feasible (score = 2.19-3.14) than doxorubicin (score = 5.81). The scores range from
15

ACCEPTED MANUSCRIPT
1 (very easy) to 10 (very difficult) based on a model with r2=0.94, which depends on analyzing
the information of already synthesized molecules and their structure complexity.
Table 5: Pharmacokinetics and medicinal chemistry parameters.
GI
absorption
High
High
High
Low
BBB
permeation
No
Pgp
substrate
No
Bioavailability
score
0.55
PAINS
alerts
1
Synthetic
Accessibility
2.34
2.34
2.3
Molecule
3a
3b
3c
3d
No
No
No
No
No
No
No
No
0.55
0.55
0.56
0.56
1
1
1
1
2.36
2.4
Low
3e
High
High
Low
No
No
No
No
No
No
0.55
0.55
0.55
1
1
1
2.53
2.57
2.5
3f
3g
3h
Low
Low
No
No
No
No
0.55
0.55
1
1
3.14
2.8
4
5
Low
Low
Low
Low
No
No
No
No
No
No
No
No
0.55
0.55
0.55
0.55
1
1
1
1
3.04
2.19
2.99
2.6
6
7
8a
8b
Low
Low
Low
Low
High
Low
High
High
High
Low
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0.55
1
1
1
1
1
1
1
1
2.68
2.81
2.76
2.49
2.5
2.81
2.63
2.63
2.9
8c
9
10a
10b
10c
11
12a
12b
12c
No
No
No
No
No
Yes
0.55
0.55
0.17
1
1
1
2.81
5.81
12d
Low
Doxorubicin
3. Conclusion
In conclusion, thiobarbiturate derivatives 3a-h, 4, 5, 6, 7, 8a-c, 9, 10a-c, 11 and 12a-d were
synthesized. Structural profile of the target compounds incorporated methylidene moiety at C5 that in
turn carried different aryl/heteroaryl groups. They were tested for anticancer activity against three
cancer cell lines e.g. Caco-2, HepG-2 and MCF-7. All the test compounds were greatly more active
than doxorubicin. Comparatively, Compound 12d was remarkably the most potent one against HepG-
2 and MCF-7. Furthermore, both a QSAR and pharmacophore model was built for these derivatives
and were highly reliable in terms of predictivity and selectivity, respectively.
4. Materials and Methods
4.1. Chemistry
All chemicals and reagents used in the current study were of analytical grade.TLC using
percolated Aluminium sheets silica gel Merck 60 F 254 and were visualized by UV lamp. Melting
points were measured in open capillary tubes using Griffin apparatus. The infrared (IR) spectra were
16

ACCEPTED MANUSCRIPT
recorded using potassium bromide disc technique on Schimadzu 435 IR Spectrophotometer Bruker
ATR/ FTIR Spectrophotometer. All the proton nuclear magnetic resonance (1HNMR) spectra and the
carbon nuclear magnetic resonance (13CNMR) spectra were performed on Varian Gemini 300 MHz
Spectrophotometer using tetramethylsilane (TMS) as internal standard. Chemical shift values (δ) are
given using parts per million scale (ppm). Mass spectra were recorded on DI-50 unit of Shimadzu GC/
MS-QP 5050A, Hewlett Packard 5988 Spectrometer and Direct Insertion Probe MS 5975 mass
selective detector. Elemental analyses (C,H,N) were performed by Micro Analytical Center, In-vitro
anticancer screening was performed by The Regional Center for Mycology and Biotechnologby, Al-
Azhar University.
4.1.1. 5-[(Dimethylamino)methylidene]-2-thioxodihydropyrimidine-4,6(1H,3H)-dione (2)
A mixture of equimolar amounts of thiobarbituric acid (0.01mol) and dimethylamidedimethyl-
acetal (0.01 mol) in dry benzene with few drops of piperidine was stirred for 5 h at room temperature.
The precipitate was filtered and crystallized from dry benzene.
Yield (95 %), mp. 180-182 °C. IR (KBr) (cm-1): 3102 (NH); 1603 (CO). 1H-NMR (DMSO-d6, D2O) δ
3.50 (s, 6H, 2CH3); 7.88 (s, 1H, CH); 11.28 (s, 2H, 2NH exchangeable with D2O). MS m/z (%): 199
(M+, 5.46); 144 (100.00). Anal. Calcd for C7H9N3O2S (199.23): C, 42.20; H, 4.55; N, 21.09%. Found:
C, 42.06; H, 4.51; N, 20.87%.
4.1.2. General procedure for the synthesis of Dihydro-5-[(arylamino)methylidene]-2-
thioxopyrimidine-4,6(1H,3H)-dione (3 a-h)
A mixture of equimolar amounts of 2 and the appropriate aromatic amine (0.01 mol) in ethanol
was refluxed for 2-5 h. The mixture was allowed to cool; the solid product was filtered and
crystallized from ethanol.
4.1.2.1. 5-[(Phenylamino)methylidene]-2-thioxo-dihydropyrimidine-4,6(1H,3H)-dion (3a)
Yield (40%), mp. 254-257 °C. IR (KBr) (cm-1): 3100 (NH); 1685 (CO). 1H-NMR (DMSO-
d6, D2O) δ 6.47-6.51 (m, 1H, 4-CH Ar); 6.52-6.55 (m, 2H, 3,5-CH Ar); 6.96-7.01 (m, 2H,
2,6-CH Ar); 8.19 (s, 1H, =CH); 10.27 (s, 3H, 3NH exchangeable with D2O). MS m/z (%):
249 (M++2, 11.01); 51 (100). Anal. Calcd for C11H9N3O2S (247.27): C, 53.43; H, 3.67; N,
16.99% Found: C, 53.64; H, 3.71; N, 17.18%.
4.1.2.2. 5-[(4-Chlorophenylamino)methylidene]-2-thioxo-dihydropyrimidine-4,6(1H,3H)-dione
(3b)
Yield (38%), mp. 251-254°C. IR (KBr) (cm-1): 3108 (NH); 1687 (CO).1H-NMR (DMSO-
d6, D2O) δ 6.53, 6.99 (2d, 4H Ar, J=6.6 Hz); 8.2 (s, 1H, =CH); 10.29 (s, 2H, 2NH
exchangeable with D2O); 11.4 (s, 1H, NH exchangeable with D2O). 13C-NMR (DMSO-d6)
δ 102.95 (C5 thiobarbituric); 115.13 (2C, C2, 6Ar); 128.41(2C, C3,5 Ar); 147.64 (C4Ar);
148.51 (C1 Ar); 163.75 (=CH-NH); 174.19 (2C, 2C=O); 176.42 (C=S). MS m/z (%): 281
(M+, 100). Anal. Calcd for C11H8ClN3O2S (281.72): C, 46.90; H, 2.86; N, 14.92% Found:
C, 47.08; H, 2.84; N, 15.13%.
17

ACCEPTED MANUSCRIPT
4.1.2.3. 5-[(4-Hydroxyphenylamino)methylidene]-2-thioxo-dihydropyrimidine-4,6(1H,3H)-
dione (3c)
Yield (68 %), mp. 226-229 °C. IR (KBr) (cm-1): 3404 (OH); 3191 (NH); 1655 (CO). H-
NMR (DMSO-d6, D2O) δ 6.79, 7.34 (2d, 4H Ar, J=9 Hz); 8.42 (s, 1H, =CH-N); 9.80 (s,
1H, OH exchangeable with D2O); 10.15 (s, 1H, NH exchangeable with D2O); 11.20 (s,
2H, 2NH exchangeable with D2O). MS m/z (%): 263 (M+, 100.00). Anal. Calcd for
C11H9N3O3S (263.27): C, 50.18; H, 3.45; N, 15.96%. Found: C, 50.32; H, 3.49; N,
16.14%.
1
4.1.2.4. 2-[(4,6-Dioxo-2-thioxo-tetrahydropyrimidin-5(6H)ylidene)methylamino]benzoic acid
(3d)
Yield (96 %), mp. 277-281 °C. IR (KBr) (cm-1): 3465 (OH); 3200 (NH); 1690 (COO);
1
1653 (CO). H-NMR (DMSO-d6, D2O) δ 6.49-6.54 (m, 1H, 5-CH Ar); 6.68 (d, 1H, 3-CH
Ar); 7.18-7.23 (m, 1H, 4-CH Ar); 7.59-7.67 (m, 1H, 6-CH Ar); 8.18 (s, 1H, =CH-N);
10.10 (s, 1H, NH exchangeable with D2O); 10.99 (s, 2H, 2NH, exchangeable with D2O);
15.98 (s, 1H, OH exchangeable with D2O). MS m/z (%): 291 (M+, 6.55); 64 (100.00).
Anal. Calcd for C12H9N3O4S (291.28): C, 49.48; H, 3.11; N, 14.43 %. Found: C, 49.62; H,
3.15; N, 14.61%.
4.1.2.5. 2-{4-[(4,6-Dioxo-2-thioxo-tetrahydropyrimidin-5(6H)-ylidene)methylamino]phenyl}-
acetic acid (3e)
Yield (43 %), mp. 296-299 °C. IR (KBr) (cm-1): 3281 (OH); 3192 (NH); 1731(COO);
1
1685 (CO). H-NMR (DMSO-d6, D2O) δ 3.59 (s, 2H, CH2); 6.56, 6.92 (2d, 4H Ar, J=8.1
Hz); 8.55 (d, 1H, =CH-N); 12.00 (s, 2H, 2NH exchangeable with D2O); 12.04 (s, 1H, NH
exchangeable with D2O); 12.13 (s, 1H, OH exchangeable with D2O). MS m/z (%):305
(M+, 25.85); 59(100.00). Anal. Calcd for C13H11N3O4S (305.31): C, 51.14; H, 3.63; N,
13.76%. Found: C, 51.28; H, 3.68; N, 13.83%.
4.1.2.6. Ethyl-4-[(4,6-dioxo-2-thioxo-tetrahydropyrimidin-5(6H)-ylidene)methylamino]benzoate
(3f)
1
Yield (47 %), mp.>300 °C. IR (KBr) (cm-1): 3199 (NH); 1708 (COO); 1680 (CO). H-
NMR (DMSO-d6, D2O) δ 1.30 (t, 3H, CH2CH3, J=7.2 Hz); 4.27 (q, 2H, CH2CH3, J=7.2
Hz); 7.67, 7.97 (2d, 4H Ar, J=8.4Hz); 8.62 (s, 1H, =CH-N); 11.37 (s, 1H, NH
exchangeable with D2O); 12.08 (s, 2H, 2NH exchangeable with D2O). 13C-NMR (DMSO-
d6) δ 14.31 (CH2CH3); 60.74 (CH2CH3); 95.04 (C5 thiobarbituric); 118.58 (2C, C2,6 Ar);
127.04 (C4 Ar); 130.71 (2C, C3,5 Ar); 142.05 (CH-NH); 151.79 (C1 Ar); 161.40 (C=O
ester); 163.72 (C=O thiobarbituric); 165.81 (C=O thiobarbituric); 177.92 (C=S). MS m/z
(%): 319 (M+, 100.00). Anal. Calcd for C14H13N3O4S (319.34): C, 52.66; H, 4.10; N, 13.16
%. Found: C, 52.89; H 4.17; N, 13.38%.
18

ACCEPTED MANUSCRIPT
4.1.2.7. 5-[(Pyridin-2-ylamino)methylen])-2-thioxo-dihydropyrimidine-4,6(1H,3H)-dione (3g)
1
Yield (54 %), mp. 222-226 °C. IR (KBr) (cm-1): 3252 (NH); 1654 (CO). H-NMR
(DMSO-d6, D2O) δ 6.41-6.48 (m, 1H, 5-CH Ar); 7.32-7.38 (m, 1H, 4-CH Ar); 7.60 (d, 1H,
3-CH Ar); 7.88 (s, 1H, 6-CH Ar); 8.20(s, 1H, =CH-N); 10.23 (s, 1H, NH exchangeable
with D2O); 11.37 (s, 2H, 2 NH exchangeable with D2O). MS m/z (%): 248 (M+, 48.50);
79 (100.00). Anal. Calcd for C10H8N4O2S (248.26): C, 48.38; H, 3.25; N, 22.57%. Found:
C, 48.5; H, 3.28; N, 22.69%.
4.1.2.8. 2-[(4,6-Dioxo-2-thioxo-tetrahydropyrimidin-5(6H)-ylidene)methylamino] isoindoline-
1,3-dione (3h)
Yield (98 %), mp. 340-341 °C. IR (KBr) (cm-1): 3227 (NH); 1656 (CO). 1H-NMR
(DMSO-d6, D2O) δ 7.86-7.93 (m, 4H Ar); 8.28 (s, 1H, =CH-N); 11.66 (s, 1H, NH
exchangeable with D2O); 11.93 (s, 2H, 2NH exchangeable with D2O). MS m/z (%): 315
(M+-1); 75 (100.00). Anal. Calcd for C13H8N4O4S (316.29): C, 49.37; H, 2.55; N, 17.71%.
Found: C, 49.62; H, 2.59; N, 17.84%.
4.1.3. General procedure for the synthesis of Bis[2-thioxo-dihydropyrimidine-4,6(1H,3H)-
dione] derivatives (4-6)
A mixture of compound 2 (0.02 mol) and appropriate aromatic diamine (0.01 mol) in ethanol
was refluxed for 3-5h. The mixture was allowed to cool and the solid product was filtered and
crystallized from ethanol.
4.1.3.1. N,N-Bis[5-methylidene-2-thioxo-dihydropyrimidine-4,6(1H,3H)-dione]piperazine (4)
1
Yield (44%), mp. 295-298 °C. IR (KBr) (cm-1): 3180 (NH); 1613 (CO). H-NMR
(DMSO-d6, D2O) δ3.07-3.09 (m, 8H, piperazine); 8.19 (s, 2H, 2 =CH-N); 11.4 (s, 4H,
4NH exchangeable with D2O). MS m/z (%): 395 (M++1, 0.24); 144 (100.00). Anal. Calcd
for C14H14N6O4S2 (394.43): C, 42.63; H, 3.58; N, 21.31 %. Found: C, 42.74; H, 3.61; N,
21.40%.
4.1.3.2. N,N-Bis[5-methylidene-2-thioxo-dihydropyrimidine-4,6(1H,3H)dione]phenylene-1,4-
diamine (5)
Yield (50 %), mp. 139-141 °C. IR (KBr) (cm-1): 3226 (NH); 1620 (CO). 1H-NMR
(DMSO-d6, D2O) δ 4.32 (s, 4H, 4NH exchangeable with D2O); 5.34 (s, 2H, 2NH
exchangeable with D2O); 6.61, 7.16 (2d, 4H Ar, J= 8.7 Hz); 8.38 (s, 2H, 2 =CH-N). MS
m/z (%): 416 (M+, 0.1); 69 (100). Anal. Calcd for C16H12N6O4S2 (416.43): C, 46.15; H,
2.90; N, 20.18% Found: C, 46.31; H, 2.93; N, 20.39%.
4.1.3.3. N,N-Bis[5-methylidene-2-thioxo-dihydropyrimidine-4,6(1H,3H)-dione]ethylene-1,2-
diamine (6)
Yield (76%), mp.>250 °C. IR (KBr) (cm-1): 3183 (NH); 1620 (CO). 1H-NMR (DMSO-d6,
D2O) δ 2.99-3.03 (m, 4H, 2CH2); 4.07(s, 2H, 2NH exchangeable with D2O); 8.08 (s, 2H, 2
=CH-N); 8.92 (s, 4H, 4NH exchangeable with D2O). MS m/z (%): 368 (M+, 43.1); 57
19

ACCEPTED MANUSCRIPT
(100.00). Anal. Calcd for C12H12N6O4S2 (368.39): C, 39.12; H, 3.28; N, 22.81%. Found:
C, 38.98; H, 3.22; N, 22.67%.
4.1.4. 5-(Hydrazinylmethylidene)-2-thioxo-dihydropyrimidine-4,6(1H,3H)-dione (7)
A mixture of of 2 (0.01 mol) and hydrazine hydrate (0.01 mol) in ethanol (10 ml) was
refluxed for 1h. The solution was allowed to cool and the precipitated solid product was filtered and
crystallized by ethanol.
Yield (93 %), mp. >320°C. IR (KBr) (cm-1): 3279, 3110 (NH, NH2); 1621 (CO). 1H-NMR
(DMSO-d6, D2O) δ 7.90 (s, 3H, NH-NH2, exchangeable with D2O); 8.03 (s, 1H, =CH-N);
9.45 (s, 2H, 2 NH exchangeable with D2O). MS m/z (%): 186 (M+, 10.12); 144 (100.00).
Anal. Calcd for C5H6N4O2S (186.19): C, 32.25; H, 3.25; N, 30.09%. Found: C, 32.39; H,
3.28; N, 30.29%.
4.1.5. General procedure for the synthesis of 5-[(3,5-Dioxopyrazolidin-1-yl)methylidene]-2-
thioxo-dihydropyrimidine-4,6(1H,3H)-dione (8a) and 5-[(5-Substituted-3-oxo-2,3-
dihydropyrazol-1-yl)methylidene]-2-thioxo-dihydropyrimidine-4,6(1H,3H)-dione (8b,c):
Equimolar amounts of 7 (0.01 mol) and the appropriate active methylidene derivative (0.01
mol) were mixed in glacial acetic acid (or dry DMF) and refluxed for 5-10 h. The mixture was allowed
to cool and the precipitated solid product was filtered and crystallized from ethanol.
4.1.5.1. 5-[(3,5-Dioxopyrazolidin-1-yl)methylidene]-2-thioxo-dihydropyrimidine-4,6(1H,3H)-
dione (8a)
1
Yield (43%), mp. >290 °C. IR (KBr) (cm-1): 3105 (NH); 1660 (CO). H-NMR (DMSO-
d6, D2O) δ 2.89 (s, 2H, CH2); 4.26 (s, 3H, 3NH exchangeable with D2O); 7.95 (s, 1H,
=CH). MS m/z (%): 254 (M+, 21.31); 52 (100). Anal. Calcd for C8H6N4O4S (254.22): C,
37.80; H, 2.38; N, 22.04% Found: C, 37.95; H, 2.42; N, 22.23%.
4.1.5.2. 5-[(5-Methyl-3-oxo-2,3-dihydropyrazol-1-yl)methylidene]-2-thioxo-dihydropyrimidine-
4,6(1H,3H)-dione (8b)
Yield (55%), mp. 283-285 °C. IR (KBr) (cm-1): 3270 (NH); 1636 (CO). 1H-NMR
(DMSO-d6, D2O) δ 2.22 (s, 3H, CH3); 5.63 (s, 1H, pyrazolyl CH); 6.95 (s, 1H, =CH-N);
11.32 (s, 2H, 2NH, exchangeable with D2O); 13.99 (s, 1H, NH, exchangeable with D2O).
13C-NMR (DMSO-d6) δ 11.23 (CH3); 89.95 (C4 pyrazolyl); 94.27 (C5 thiobarbituric);
145.18 (=CH-NH); 151.45 (C5 pyrazolyl); 158.00 (C=O pyrazolyl); 161.90 (C=O
thiobarbituric); 163.00 (C=O thiobarbituric); 173.96 (C=S). MS m/z (%): 253 (M+1, 2.31);
198 (100). Anal. Calcd for C9H8N4O3S (252.25): C, 42.85; H, 3.20; N, 22.21%. Found: C,
42.91; H, 3.23; N, 22.31%.
4.1.5.3. 5-[(5-Amino-3-oxo-2,3-dihydropyrazol-1-yl)methylidene]-2-thioxo-dihydropyrimidine-
4,6(1H,3H)-dione (8c)
1
Yield (38%), mp. >320 °C. IR (KBr) (cm1): 3278 (NH); 3184 (NH2); 1650 (CO). H-
NMR (DMSO-d6, D2O) 7.47 (s, 1H, CH pyrazolyl); 8.25 (s, 1H, =CH-N); 11.31 (s, 2H,
20

ACCEPTED MANUSCRIPT
NH2, exchangeable with D2O); 11.59 (s, 1H, NH, exchangeable with D2O); 11.88 (s, 2H,
2NH, exchangeable with D2O). MS m/z (%): 254 (M++1, 13.69); 99 (100). Anal. Calcd
for C8H7N5O3S (253.24): C, 37.94; H, 2.79; N, 27.66%. Found: C, 37.87; H, 2.78; N,
27.52%.
4.1.6. N,N-Bis(5-methylidene-2-thioxo-dihydropyrimidine-4,6(1H,3H)-dione)hydrazine (9)
A mixture of equimolar amounts of 2and the hydrazide derivative 7 (0.01 mol) was refluxed in
ethanol for 5h. The mixture was allowed to cool and the solid product was filtered and crystallized by
1
ethanol. Yield (69%), mp. >300 °C. IR (KBr) (cm-1): 3273 (NH); 1612 (CO). H-NMR (DMSO-d6,
D2O) δ 5.83(s, 2H, 2NH exchangeable with D2O); 8.03 (s, 2H, 2 =CH-N); 8.39 (s, 4H, 4NH
exchangeable with D2O). MS m/z (%): 337 (M+-3, 1.19); 108 (100). Anal. Calcd for C10H8N6O4S2
(340.34): C, 35.29; H, 2.37; N, 24.69% Found: C, 35.38; H, 2.36; N, 24.82%.
4.1.7. General procedure for the synthesis of 4-Bromo-N'-[(4,6-dioxo-2-thioxo-
tetrahydropyrimidin-5(6H)-ylidene)methyl]benzenesulfonohydrazide (10a) N'-[(4,6-
Dioxo-2-thioxo-tetrahydropyrimidin-5(6H)-ylidene)methyl]benzohydrazide derivatives
(10b, 10c)
A mixture of equimolar amounts of 7 (0.01 mol) and p-bromobenzenesulfonylchloride (0.01
mol) or benzoyl chloride (0.01 mol) or p-chlorobenzoyl chloride (0.01 mol) was refluxed in ethanol
for 5 h to yield compounds 10a, 10b and 10c, respectively. The mixture was allowed to cool and the
solid product was filtered and crystallized first from ethanol followed by hot water.
4.1.7.1. 4-Bromo-N'-[(4,6-dioxo-2-thioxo-tetrahydropyrimidin-5(6H)-ylidene)methyl]benzene-
sulfonohydrazide (10a)
Yield (39%), mp. 223-225 °C. IR (KBr) (cm-1): 3189 (NH); 1689 (CO); 1343, 1163
(SO2). 1H-NMR (DMSO-d6, D2O) δ 7.68 -7.76 (m, 5H, 4 Ar H+ 1 =CH); 10.03-10.16 (m,
4H, 4NH, exchangeable with D2O). MS m/z (%): 407 (M++2, 1.47); 69 (100.00). Anal.
Calcd for C11H9BrN4O4S2 (405.25): C, 32.60; H, 2.24; N, 13.83%. Found: C, 32.71; H,
2.25; N, 14.01%.
4.1.7.2. N'-[(4,6-Dioxo-2-thioxo-tetrahydropyrimidin-5(6H)-ylidene)methyl]benzohydrazide
(10b)
Yield (42%), mp. >340 °C. IR (KBr) (cm-1): 3270 (NH); 1630 (CO). 1H-NMR (DMSO-d6,
D2O) δ 7.50-7.58 (m, 2H, 3,5-CH Ar); 7.61-7.63 (m, 1H, 4-CH Ar); 7.89-7.94 (m, 2H, 2,6-
CH Ar); 8.28 (s, 1H, =CH-N); 10.62 (s, 1H, NH-CH exchangeable with D2O); 11.38 (s,
1H, NH-CO exchangeable with D2O); 11.95 (s, 2H, 2NH exchangeable with D2O). MS
m/z (%): 290 (M+, 2.51); 105 (100). Anal. Calcd for C12H10N4O3S (290.3): C, 49.65; H,
3.47; N, 19.30%. Found: C, 49.79; H, 3.50; N, 19.49%.
21

ACCEPTED MANUSCRIPT
4.1.7.3. 4-Chloro-N'-[(4,6-dioxo-2-thioxo-tetrahydropyrimidin-5(6H)-
ylidene)methyl]benzohydrazide (10c)
Yield (33%), mp. >300 °C. IR (KBr) (cm-1): 3172 (NH); 1641(CO).1H-NMR (DMSO-d6,
D2O) δ 5.58 (s, 2H, 2NH, exchangeable with D2O); 7.53 (s, 1H, =CH-N); 7.60, 7.92 (2d,
4H Ar, J=7.2Hz); 10.66 (s, 2H, 2NH exchangeable with D2O). MS m/z (%): 323 (M+-1,
4.16); 299 (100). Anal. Calcd for C12H9ClN4O3S (324.74): C, 44.38; H, 2.79; N, 17.25%.
Found: C, 44.60; H, 2.83; N, 17.48%.
4.1.8. 1-[(Tetrahydro-4,6-dioxo-2-thioxopyrimidin-5(6H)-ylidene)methyl]-4-
phenylthiosemicarbazide (11)
A mixture of equimolar amounts of 7 (0.01 mol) and phenyl isothiocyanate (0.01 mol) in
ethanol was refluxed for 5 h. The mixture was allowed to cool and the solid product was filtered and
crystallized from ethanol. Yield (64%), mp. 152-155 °C. IR (KBr) (cm-1): 3302, 3201 (NH); 1669
1
(CO). H-NMR (DMSO-d6, D2O) δ: 4.26 (s, 2H, NH-NH exchangeable with D2O); 4.42 (s, 1H, NH-
CS exchangeable with D2O); 7.05-7.10 (m, 1H, 4-CH Ar); 7.32-7.39 (m, 2H, 3,5-CH Ar); 7.58-7.65
(m, 2H, 2,6-CH Ar); 8.39 (s, 1H, =CH); 9.03 (s, 2H, 2NH exchangeable with D2O). MS m/z (%): 322
(M++1, 0.25); 50 (100). Anal. Calcd for C12H11N5O2S2 (321.38): C, 44.85; H, 3.45; N, 21.79%. Found:
C, 44.97; H, 3.48; N, 21.97%.
4.1.9. General procedure for the synthesis of 5-[(2-Benzylidenehydrazinyl)methylidene]-2-
thioxo-dihydropyrimidine-4,6(1H,3H)-dione derivatives (12a-c) 5-[(2-(Thiophen-2-
ylmethylidene)hydrazinyl)methylidene]-2-thioxo-dihydropyrimidine-4,6(1H,3H)-dione
(12d)
A mixture of equimolar amounts of 7 (0.01 mol) and the appropriate aromatic aldehyde (0.01
mol) in ethanol was refluxed for 1-3 h. The mixture was allowed to cool and the solid product was
filtered and crystallized from ethanol.
4.1.9.1. 5-[(2-Benzylidenehydrazinyl)methylidene]-2-thioxo-dihydropyrimidine-4,6(1H,3H)-
dione (12a)
Yield (55%), mp. >280 °C. IR (KBr) (cm-1): 3122 (NH); 1655 (CO). 1H-NMR (DMSO-d6,
D2O) δ 7.46 (s, 1H, =CH-N); 7.48-7.56 (m, 3H, 3,4,5-CH Ar); 8.11 (d, 2H, 2,6-CH Ar);
8.29 (s, 1H, N=CH); 12.31 (s, 1H, NH exchangeable with D2O); 12.43 (s, 2H, 2NH
exchangeable with D2O). 13C-NMR (DMSO-d6) δ 119.12 (C5 thiobarbituric); 128.08 (2C,
C3, 5Ar); 132.63 (C1 Ar); 133.37 (3C, C2,4,6 Ar); 155.56 (C=N); 159.35 (=C-N); 161.62
(2C, 2C=O); 178.56 (C=S). MS m/z (%): 274 (M+, 2.57); 135 (100). Anal. Calcd for
C12H10N4O2S (274.3): C, 52.54; H, 3.67; N, 20.43%. Found: C, 52.82; H, 3.73; N, 20.67%.
4.1.9.2. 5-[(2-(4-Methoxybenzylidene)hydrazinyl)methylidene]-2-thioxo-dihydro pyrimidine-
4,6(1H,3H)-dione (12b)
Yield (58%), mp. >250 °C. IR (KBr) (cm-1): 3254 (NH); 1640 (CO). 1H-NMR (DMSO-d6,
D2O) δ 3.74 (s, 3H, CH3); 6.70, 6.88 (2d, 4H Ar, J=8.7 Hz); 7.42 (d, 1H, =CH-N); 7.65 (s,
1H, N=CH); 11.5 (s, 3H, 3NH exchangeable with D2O). MS m/z (%): 304 (M+, 12.19);
22

ACCEPTED MANUSCRIPT
164 (100). Anal. Calcd for: C13H12N4O3S (304.32): C, 51.31; H, 3.97; N, 18.41%. Found:
C, 51.48; H, 4.02; N, 18.53%.
4.1.9.3. 5-[(2-(2,3-dimethoxybenzylidene)hydrazinyl)methylidene]-2-thioxo-dihydropyrimidine-
4,6(1H,3H)-dione (12c)
Yield (90%), mp. 278-279 °C. IR (KBr) (cm-1): 3238 (NH); 1625 (CO). 1H-NMR
(DMSO-d6, D2O) δ 3.80,3.82 (2s, 6H, 2 OCH3); 7.07-7.18 (m, 1H, 4-CH Ar); 7.42-7.45
(m, 1H, 5-CH Ar); 7.95-7.98 (m, 1H, 6-CH Ar); 8.47 (s, 1H, =CH-N); 8.93 (s, 1H,
N=CH); 10.35 (s, H, NH exchangeable with D2O); 12.55 (s, 2H, 2NH exchangeable with
D2O). MS m/z (%): 334 (M+, 3.28); 149 (100.00). Anal. Calcd for C14H14N4O4S (334.35):
C, 50.29; H, 4.22; N, 16.76%. Found: C, 50.38; H, 4.25; N, 16.89%.
4.1.9.4. 5-[(2-(thiophen-2-ylmethylidene)hydrazinyl)methylidene]-2-thioxo-dihydropyrimidine-
4,6(1H,3H)-dione (12d)
Yield (60 %), mp. >320 °C. IR (KBr) (cm-1): 3181 (NH); 1669 (CO).1H-NMR (DMSO-
d6, D2O) δ 4.32(s, 1H, NH exchangeable with D2O); 7.12 (t, 1H, 4-CH thiophene); 7.46 (d,
1H, 3-CH thiophene); 7.62 (d, 1H, 5-CH thiophene); 7.99 (s, 1H, =CH-N); 8.41 (s, 1H,
N=CH); 10.98 (s, 2H, 2NH exchangeable with D2O). MS m/z (%): 280 (M+, 0.53); 238
(100.00). Anal. Calcd for C10H8N4O2S2 (280.33): C, 42.85; H, 2.88; N, 19.99%. Found: C,
42.99; H, 2.90; N, 20.07%.
4.2. Antitumor activity
The in-vitro anti-tumor activity of the test compounds was performed in the Regional Center
for Mycology and Biotechnology, Al Azhar University. The antiproliferative activities of the prepared
compounds against colorectal cancer (Caco-2), hepatic cancer (HepG-2) and breast cancer (MCF-7)
cell lines were evaluated using SRB method as described by Skehan et al., 1990[38]. Exponentially
growing cells were collected using 0.25% Trypsin-EDTA and seeded in 96-well plates at 1000-2000
cells/well in RPMI-1640 supplemented medium. After 24 hr, cells were incubated for 72 hr with
various concentrations of the tested compounds. Following 72 hr treatment, the cells were fixed with
10% trichloroacetic acid for 1 h at 4 ºC. Wells were stained for 10 min at room temperature with 0.4%
SRB dissolved in 1% acetic acid. The plates were air dried for 24 h and the dye was solubilized with
Tris-HCl for 5 min on a shaker at 1600 rpm. The optical density (OD) of each well was measured
spectrophotometrically at 564 nm with an ELISA microplate reader (ChroMate-4300, FL, USA). The
IC50 values were calculated according to the equation for Boltzman sigmoidal concentration–response
curve using the nonlinear regression fitting models (Graph Pad, Prism Version 5).
4.3. QSAR
As an initial step before working on the QSAR model fetching, the cytotoxic activity of the 24
compounds for breast cancer cell line (MCF-7) were presented as PIC50 (-log (IC50*10-6)). Next, the
compounds were classified into classified into two sets with ratio 3:1, training set including 18
compounds and test set including the other 6 compounds. To assure random classification and
structural diversity, the test set includes at least one compound from each synthesis scheme. Many 2D
23

ACCEPTED MANUSCRIPT
QSAR models have been built on the training set using Molecular Operating Environment (MOE
2016)[32] with partial least squares (PLS) as model fitting procedure and examined on the test set
compounds. The best one was chosen according to statistical values that assures the good prediction
ability of the model internally and externally, as r2 (correlation coefficient for training set of
compounds) and pred_r2 (predictive r2 for the test set of compounds). The 2D descriptors were filtered
[33-34]
using WEKA 3.9
and contingency analysis in MOE [32] to select the most relevant ones. Then,
the resultant descriptors were tried by mix and match to form different models.
4.4. Pharmacophore
A ligand based pharmacophore model was built with regards to the MCF-7 breast cancer cell
line cytotoxicity results using MOE 2016 based on flexible alignment of 15 structurally diverse
compounds from the different synthesis schemes. The pharmacophore consensus threshold score was
first set to 100% and there were no features available. Then, it was reduced to 97% which yielded only
3 feature and finally it was adjusted to 93% which resulted in 17 features. The best model was
validated internally and externally.
4.5. In silico physicochemical and ADME properties prediction
The molecular structures were converted into SMILES database using MOE2016. Then, these
SMILES were inserted as input in SwissADME website to calculate the physicochemical descriptors,
pharmacokinetics properties, ADME parameters and medicinal chemistry friendliness.
Acknowledgement
The corresponding author would like to acknowledge ASRT for providing the funding grant
ASRT-4/2016 part of which was used for partial support of this research work.
Conflict of Interest
The authors declare no conflict of interest.
24

ACCEPTED MANUSCRIPT
References
[1] J.J. Dimaggio, R.P. Warrell, J. Muindi, Y.-W. Stevens, S.J. Lee, D.A. Lowenthal, I. Haines,
T.D. Walsh, L. Baltzer, S. Yaldaei, C.W. Young, Phase I Clinical and Pharmacological Study
of Merbarone, Cancer Res., 1990, 50, 1151-1155.
[2] J.M. Fortune, N. Osheroff, Merbarone Inhibits the Catalytic Activity of Human Topoisomerase
IIα by Blocking DNA Cleavage, J. Biol. Chem. 1998, 273, 17643-17650.
[3] A.K. Larsen, A.E. Escargueil, A. Skladanowski, Catalytic topoisomerase II inhibitors in cancer
therapy, Pharmacol. Ther., 2003, 99, 167-181.
[4] A. Ranise, A. Spallarossa, S. Schenone, O. Bruno, F. Bondavalli, A. Pani, M.E. Marongiu, V.
Mascia, P. La Colla, R. Loddo, Synthesis and antiproliferative activity of basic thioanalogues
of merbarone, Bioorg. Med. Chem., 2003, 11, 2575-2589.
[5] Y. Qin, L. Meng, C. Hu, W. Duan, Z. Zuo, L. Lin, X. Zhang, J. Ding, Gambogic acid inhibits
the catalytic activity of human topoisomerase IIα by binding to its ATPase domain, Mol.
Cancer Ther., 2007, 6, 2429-2440.
[6] J. Schemies, W. Sippl, M. Jung, Histone deacetylase inhibitors that target tubulin, Cancer Lett.
2009, 280, 222-232.
[7] Y. Cen, Sirtuins inhibitors: The approach to affinity and selectivity: a review, Biochim.
Biophys. Acta, 2010, 1804, 1635-1644.
[8] P. Sivaraman, S. Mattegunta, G.V. Subbaraju, C. Satyanarayana, B. Padmanabhan, Design of a
novel nucleoside analog as potent inhibitor of the NAD dependent deacetylase, SIRT2, Syst.
Synth. Biol., 2011, 4, 257-263.
[9] X. Kong, J. Qin, Z. Li, A. Vultur, L. Tong, E. Feng, G. Rajan, S. Liu, J. Lu, Z. Liang, M.
Zheng, W. Zhu, H. Jiang, M. Herlyn, H. Liu, R. Marmorstein, C. Luo, Development of a novel
class of B-Raf(V600E)-selective inhibitors through virtual screening and hierarchical hit
optimization, Org. Biomol. Chem., 2012, 10, 7402-7417.
[10]
S. Bruzzone, M.D. Parenti, A. Grozio, A. Ballestrero, I. Bauer, A. Del Rio, A.
Nencioni, Rejuvenating sirtuins: the rise of a new family of cancer drug targets, Curr. Pharm.
Des., 2013, 19, 614-623.
[11]
N. R. Penthala, P. R. Ponugoti, V. Kasam, P. A. Crooks, 5-((1-Aroyl-1H-indol-3-
yl)methylidene)-2-thioxodihydropyrimidine-4,6(1H,5H)-diones as potential anticancer agents
with anti-inflammatory properties, Bioorg. Med. Chem. Lett., 2013, 23, 1442-1446.
[12]
P. Bhatt, M. Kumar, A. Jha, Design, Synthesis and Anticancer Evaluation of
Oxa/Thiadiazolylhydrazones of Barbituric and Thiobarbituric Acid: A Collective In Vitro and
In Silico Approach, Chemistry Select, 2018, 3, 7060-7065.
[13]
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, Eur. J. Med. Chem., 2018, 143, 1919-1930.
[14]
J. Figueiredo, , J. L. Serrano, , E. Cavalheiro, , L. Keurulainen, , J. Yli-Kauhaluoma, ,
V. M. Moreira, S. Ferreira, F.C. Domingues, S. Silvestre, P. Almeida,. Trisubstituted
barbiturates and thiobarbiturates: Synthesis and biological evaluation as xanthine oxidase
inhibitors, antioxidants, antibacterial and anti-proliferative agents, Eur. J. Med. Chem., 2018,
143, 829-842.
[15] D.C. Young, T.R. Bailey, Methods for treating or preventing viral infections and
associated diseases, US Patent 9920402, 2001.
[16]
S. Rajamaki, A. Innitzer, C. Falciani, C. Tintori, F. Christ, M. Witvrouw, Z. Debyser, S.
Massa, M. Botta, Exploration of novel thiobarbituric acid-, rhodanine- and thiohydantoin-
based HIV-1 integrase inhibitors, Bioorg. Med. Chem. Lett., 2009, 19, 3615-3618.
25

ACCEPTED MANUSCRIPT
[17]
J. H. Lee, S. Lee, M. Y. Park, H. J. Ha, H. Myung, Evaluation of inhibitory effects of
thiobarbituric acid derivatives targeting HCV NS5B polymerase, J. Microbiol. Biotechnol.,
2010, 20, 510-512.
[18]
G. S. Hassan, N. A. Farag, G. H. Hegazy, R. K. Arafa, Design and Synthesis of Novel
Benzopyran‐ 2‐ one Derivatives of Expected Antimicrobial Activity through DNA Gyrase‐ B
Inhibition. Arch. Pharm., 2008, 341, 725-733.
[19]
F. A. Ragab, T. A. A. Yahya, M. M. El-Naa, R. K. Arafa, Design, synthesis and
structure–activity relationship of novel semi-synthetic flavonoids as antiproliferative agents,
Eur. J. Med. Chem., 2014, 82, 506-520.
[20]
M. A. Ismail, R. K. Arafa, M. M. Youssef, W. M. El-Sayed, Anticancer, antioxidant
activities, and DNA affinity of novel monocationic bithiophenes and analogues, Drug Des.
Dev. Ther., 2014, 8, 1659-1672.
[21]
N. E. Abo-Elmagd, R. F. George, M. A. Ezzat, R. K. Arafa, New 1-phthalazinone
scaffold based compounds: Design, synthesis, cytotoxicity and protein kinase inhibition
activity, Mini-Rev. Med. Chem., 2018, 18, 1759 - 1774.
[22]
[23]
U. Pindur, N,N-Dimethylformamide Diethyl Acetal, e-EROS (2001).
F. A. Abu-Shanab, S. A. S. Mousa, E. A. Eshak, A. Z. Sayed, A. Al-Harrasi,
Dimethylformamide Dimethyl Acetal (DMFDMA) in Heterocyclic Synthesis: Synthesis of
Polysubstituted Pyridines, Pyrimidines, Pyridazine and Their Fused Derivatives, Int. J. Org.
Chem., 2011, 1, 207-214.
[24]
D. Kumar, V. Judge, R. Narang, S. Sangwan, E. De Clercq, J. Balzarini, B.
Narasimhan, Benzylidene/2-chlorobenzylidene hydrazides: synthesis, antimicrobial activity,
QSAR studies and antiviral evaluation, Eur. J. Med. Chem., 2010, 45, 2806-2816.
[25]
M. I. El-Zahar, S. S. Abd El-Karim, M. E. Haiba, Synthesis and Cytotoxic Evaluation
of Some Novel 6-(Benzofuran-2-yl)-4-(4-Fluorophenyl)Pyridines, World J. Chemistry, 2009,
4, 182-194
[26]
M. M. Abd El-All, A. H. Halawa, A. A. Hassan, M. A. El-Nassag, G. A. Abd El-Jaleel,
E. M. Eliwa, A. H. Bedair, Synthesis and Antioxidant Activity of Some Derivatives of 2-(2-
oxo-4-phenyl-2h-chromen-7-yloxy)AcetoHydrazide, J. Atoms Mol., 2013, 3, 537-552.
[27]
W.S. El-Hamouly, K. M. Amin, S. A. El-Assaly, E. A. Abd El-Meguid, Synthesis and
antitumor activity of some new N-substituted-sulfonyl, 1,2,4-triazole, N-substituted-
benzylidene and pyrrole derivatives attached to 4-(benzo[d]thiazol-2-yl)benzohydrazide, Der
pharma chem., 2011, 3, 293-306.
[28] A. H. Sharba, R. H. Al-Bayati, M. Aouad, N. Rezki, Synthesis of oxadiazoles,
thiadiazoles and triazoles derived from benzo[b]thiophene, Molecules, 2005, 10, 1161-1168.
[29]
P. Kumar, B. Narasimhan, D. Sharma, Substituted benzoic acid benzylidene/furan-2-yl-
methylidene hydrazides: synthesis, antimicrobial evaluation and QSAR analysis, Arkivoc,
2008, 13, 159-178.
[30]
L. Srikanth, U. Naik, R. Jadhav, N. Raghunandan, J. V. Rao, Synthesis and Evaluation
of New Phenylaminothiadiazolo-Oxadiazolo-1,3benzoxazoles for Their Antibacterial Activity,
Int. J. Pharm. Biol. Sci. 2010, 1, 260-271.
[31]
[32]
[33]
H. A. Abdel-Aziz, T. Elsaman, M.I. Attia, A. M. Alanazi, The reaction of ethyl 2-oxo-
2H-chromene-3-carboxylate with hydrazine hydrate, Molecules, 2013, 18, 2084-2095.
Molecular Operating Environment (MOE), 2016.08; Chemical Computing Group Inc.,
1010 Sherbrooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2016.
M. Hall, E. Frank, G. Holmes, B. Pfahringer, P. Reutemann, I. H. Witten, The WEKA
data mining software: an update. ACM SIGKDD explorations newsletter, 2009, 11, 10-18.
26

ACCEPTED MANUSCRIPT
[34]
E. Frank, M. A. Hall, I. H. Witten (2016). The WEKA Workbench. Online Appendix
for "Data Mining: Practical Machine Learning Tools and Techniques", Morgan Kaufmann,
Fourth Edition.
[35]
[36]
[37]
RNA Targeted Library http://www.otavachemicals.com/products/targeted-libraries-
and-focused-libraries/rna-binding (last accessed Oct 24, 2018).
SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and
medicinal chemistry friendliness of small molecules. Sci. Rep., 2017, 7, 42717.
S. J. Capuzzi, E. N. Muratov, A.Tropsha, Phantom PAINS: Problems with the Utility of
Alerts for Pan-Assay INterference CompoundS, J. Chem. Inf. Model., 2017, 57, 417–427.
[38]
P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, J. T.
Warren, H. Bokesch, S. Kenney, M. R. Boyd, New colorimetric cytotoxicity assay for
anticancer-drug screening. J. Natl. Cancer Inst., 1990, 82, 1107-1112.
27

ACCEPTED MANUSCRIPT
Legends
List of figures:
Figure 1:
Figure 2:
Merbarone I and thiobarbiturate analogues with promising anticancer activity.
Strucutral design of the target thiobarbiturate derivatives. Fragment replacement profile of
reported thiobarbiturates (V, VI) and designed thiobarbituric acid derivatives as antitumor. First
modification (upper panel) in compounds 2, 3a-h, 7, 10a-c, 11 and 12a-d, moiety B was
replaced by different amines e.g. amino group, hydrazone, hydrazinocarbonyl,
hydrazinosulfone and thiosemicarbazide moieties. Second modification in compounds 8a-c
(middle panel) explored isosteric replacement of furan ring by pyrazole moiety.
Thirdmodification (lower panel) for compounds 4-6 and 9 included replacing moiety B in V
and VI with aliphatic/aryl diamine linker to obtain bis-thiobarbiturates.
Figure 3:
Figure 4:
Figure 5:
Figure 6:
Correlation plot of the training set showing R2 value = 0.755
Correlation plot of the test set showing R2 value= 0.704
Five features of the pharmacophore model on the 15 aligned compounds
3D spatial distribution of the five pharmacophoric features
List of tables:
Table 1:
IC50 of the test set of compounds against human colorectal cancer (Caco-2), hepatic cancer
(HepG-2), and breast cancer (MCF-7).
Table 2:
Table 3:
Compounds in the training set with their pIC50 values (MCF-7 cell line), predicted pIC50,
residuals and Z-scores
Compounds in the test set with their PIC50 values (MCF-7 cell line), predicted pIC50 and the
residual values.
Table 4:
Table 5:
Drug-likeness based on Lipinski’s rule of five, TPSA and number of rotatable bonds
Pharmacokinetics and medicinal chemistry parameters
List of schemes:
Scheme 1:
Scheme 2:
Scheme 3:
Scheme 4:
Scheme 5:
Synthesis of derivatives 3a-h.
Synthesis of derivatives 4-6.
Synthesis of derivatives 7 and 8a-c.
Synthesis of derivative 9.
Synthesis of derivatives 10a-c, 11 and 12a-d.
List of abbreviations:
PEOE:
VSA:
Partial Equalization of Orbital Electronegativities [Gasteiger 1980]
Van der waals surface area
Burden cas university of texas descriptors using adjacency martix
Molecular refractivity
BCUT:
SMR:
FNEG:
QSAR:
XRMSE:
TPSA:
Mw:
Fractional Negative
Quantitative structure activity relationship
Cross root mean square error
Total polar surface area
Molecular weight
2D:
Two dimensional
PiR:
Pi ring center
28

ACCEPTED MANUSCRIPT
Aro:
Hyd:
Acc:
Don:
IR:
Aromatic
Hydrophobic
H-bond acceptor
H-bond donor
Infrared spectroscopy
Mass spectrometer
Nuclear magnetic resonance
MS:
NMR:
29