Design, synthesis, and bioactivity of dihydropyrimidine derivatives as kinesin spindle protein inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.115126

A series of twenty-one 3,4-dihydropyrimidine derivatives bearing the heterocyclic 1,3-benzodioxole at position 4 in addition to different substituents at positions 2, 3 and 5 were designed and synthesized as monastrol analogs. The novel synthesized compounds were screened for their cytotoxic activity towards 60 cancer cell lines according to NCI (USA) protocol. Compounds 10b and 15 showed the best antitumor activity against most cell lines. Compound 15 was subsequently tested in 5-doses mode and displayed high selectivity towards CNS, prostate and leukemia subpanel with selectivity ratios of 22.30, 15.38 and 12.56, respectively at GI₅₀ level. The IC₅₀ of compounds 9d, 10b, 12, 15 and 16 against kinesin enzyme were 3.86 ± 0.12, 10.70 ± 0.35, 3.95 ± 0.12, 4.36 ± 0.14, and 14.07 ± 0.45 μM respectively, while the prototype compound, monastrol, reported IC₅₀ value of 20 ± 0.42 μM. The safest compound among test compounds against normal cell line (HEK 293) is 10b with IC₅₀ value of 62.02 ± 2.42 μM/ml in comparison to doxorubicin (IC₅₀ = 11.34 ± 0.44 μM/ml). Cell cycle analysis of SNB-75 cells treated with compound 15 showed cell cycle arrest at G2/M phase. Further, the assay of levels of active caspase-3 and caspase-9 was investigated. Moreover, Molecular docking of compounds, 9d, 10b, 12, 15, 16, monastrol and mon-97 was performed to study the interaction between inhibitors and the kinesin spindle protein allosteric binding site.

Full text of DOI:10.1016/j.bmc.2019.115126

Journal Pre-proofs
Design, synthesis, and bioactivity of dihydropyrimidine derivatives as kinesin
spindle protein inhibitors
Haytham O. Tawfik, Mervat H. El-Hamamsy, Nabaweya A. Sharafeldin,
Tarek F. El-Moselhy
PII:
S0968-0896(19)31159-9
https://doi.org/10.1016/j.bmc.2019.115126
BMC 115126
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
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Revised Date:
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9 July 2019
12 September 2019
18 September 2019
Please cite this article as: H.O. Tawfik, M.H. El-Hamamsy, N.A. Sharafeldin, T.F. El-Moselhy, Design,
synthesis, and bioactivity of dihydropyrimidine derivatives as kinesin spindle protein inhibitors, Bioorganic &
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.115126
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Design, synthesis, and bioactivity of dihydropyrimidine derivatives as kinesin
spindle protein inhibitors
Haytham O. Tawfik, Mervat H. El-Hamamsy, Nabaweya A. Sharafeldin, and Tarek F. El-Moselhy
1. Haytham O. Tawfik, MSc
Assistant Lecturer, Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Tanta University, El Giesh street, Tanta, 31527, Egypt.
Email: haytham.omar.mahmoud@pharm.tanta.edu.eg
Tel. +201005356819; Fax: +20403335466
2. Mervat H. El-Hamamsy, PhD
Associate Professor, Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Tanta University, El Giesh street, Tanta, 31527, Egypt.
Email: mhamamsy@pharm.tanta.edu.eg
Tel. +201008121831
3. Nabaweya A. Sharafeldin, PhD
Associate Professor, Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Tanta University, El Giesh street, Tanta, 31527, Egypt.
Email: nabawia.eldeen@pharm.tanta.edu.eg
Tel. +201022489700
4. Tarek F. El-Moselhy, PhD
Professor and Chairman of the Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Tanta University, El Giesh street, Tanta, 31527, Egypt.
Email: tarek.faathy@pharm.tanta.edu.eg
Tel. + 201006358839
1

Design, synthesis, and bioactivity of dihydropyrimidine derivatives as kinesin
spindle protein inhibitors
Haytham O. Tawfik*, Mervat H. El-Hamamsy, Nabaweya A. Sharafeldin and Tarek F. El-Moselhy
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Tanta University,
Tanta, 31527, Egypt.
*Corresponding author: H. O. Tawfik, E-mail: haytham.omar.mahmoud@pharm.tanta.edu.eg
Tel: +201005356819; Fax: +2040-3335466
Abstract
A series of twenty-one 3,4-dihydropyrimidine derivatives bearing the heterocyclic 1,3-benzodioxole
at position 4 in addition to different substituents at positions 2, 3 and 5 were designed and synthesized
as monastrol analogs. The novel synthesized compounds were screened for their cytotoxic activity
towards 60 cancer cell lines according to NCI (USA) protocol. Compounds 10b and 15 showed the
best antitumor activity against most cell lines. Compound 15 was subsequently tested in 5-doses mode
and displayed high selectivity towards CNS, prostate and leukemia subpanel with selectivity ratios
of 22.30, 15.38 and 12.56, respectively at GI50 level. The IC50 of compounds 9d, 10b, 12, 15 and 16
against kinesin enzyme were 3.86 ± 0.12, 10.70 ± 0.35, 3.95 ± 0.12, 4.36 ± 0.14, and 14.07 ± 0.45
μM respectively, while the prototype compound, monastrol, reported IC50 value of 20 ± 0.42 μM.
The safest compound among test compounds against normal cell line (HEK 293) is 10b with IC50
value of 62.02 ± 2.42 µM/ml in comparison to doxorubicin (IC50 = 11.34 ± 0.44 µM/ml). Cell cycle
analysis of SNB-75 cells treated with compound 15 showed cell cycle arrest at G2/M phase. Further,
the assay of levels of active caspase-3 and caspase-9 was investigated. Moreover, Molecular docking
of compounds, 9d, 10b, 12, 15, 16, monastrol and mon-97 was performed to study the interaction
between inhibitors and the kinesin spindle protein allosteric binding site.
Keywords: Dihydropyrimidine; kinesin spindle protein; cell cycle analysis; apoptosis; molecular
docking.
1. Introduction
Kinesins are class of motor proteins that responsible for many central purposes inside the cell [1, 2].
Generally, these proteins bind to microtubules and use the energy resulting from ATP hydrolysis to
complete their roles. The mitotic kinesin, a subclass of kinesin, is expressed in cell division at mitotic
phase to establish the mitotic spindle, and other roles [3]. One of the features of cancer cells is
uncontrolled cell proliferation. The classical chemotherapeutic drug like taxanes (paclitaxel) and
Vinca alkaloids (vincristine, vinblastine), that interfering with microtubule dynamics, are still as
anticancer agents used in the clinic [4, 5]. However, due to the presence of microtubules in non-
dividing cells such as, post-mitotic neurons, unwanted side effects, like peripheral neuropathy, are
2

often detected in patients treated with these drugs [6]. The mitotic kinesin family consists of more
than 10 members, which show either unique or overlapping roles in mitosis [7]. Kinesin spindle
protein (KSP, or Eg5), is considered the most dramatic target protein of the mitotic kinesin family.
KSP contains a catalytic ATPase activity that provides energy for directed movement along
microtubules. Therefore, inhibition of ATPase activity will trouble the role of KSP effectively. Most
of KSP inhibitors are ATP-uncompetitive allosteric inhibitors that inhibit ATPase activity reversibly
and include diverse of structural categories. Recently, many KSP inhibitors have entered pre-clinical
and clinical trials as anticancer agents and have revealed satisfying effect with IC50 of sub-nanomolar
to low nanomolar levels [8]. From monastrol having dihydropyrimidine nucleus, the earliest found
KSP inhibitor but did not enter clinical trial, passing by s-trityl-l-cysteine (STLC), dihydropyrroles
and dihydropyrazoles (e.g. MK-0731, Arry-520), which have entered phase I and II clinical trials, to
quinazolines (e.g. ispinesib) which has entered a phase II clinical trial, KSP is still promising target
for designing new anticancer agents [9].
In 1999, Mayer et al, discovered monastrol (Figure 1) as the first small molecule able to inhibit
several targets as anticancer including; the mitotic kinesin Eg5 (KSP) [10-12], 5-lipoxygenase
enzyme [13], mTOR [14], and cell division cycle 25 (CDC25) phosphatases [15]. Monastrol causes
mitotic arrest at G2/M phase by blocking bipolar mitotic spindle in mammalian cells leading to cell
apoptosis [16, 17]. Monastrol has motivated the medicinal chemists to utilize structure-based drug
design methods to design new antimitotic agents through structural manipulation of the
dihydropyrimidine (DHPM) scaffolds to generate novel compounds for cancer therapy. [9, 18]
2. Rationale and Design
Dihydropyrimidine (DHPM) derivatives were synthesized by Biginelli [19] through multi-component
reaction. Many DHPM-based derivatives have presented important biological functions including
antitumor and other biological activities [20]. Since the discovery of monastrol (IC50 = 20 ± 0.42
μM) (Figure 1A) as a weak antimitotic anticancer DHPM derivative, many attempts have been
reported to develop more potent inhibitors of kinesin Eg5 based on monastrol scaffold [21-23].
Piperastrol (Figure 1A), the 3,4-methylenedioxy analogue of monastrol, (IC50 = 6.6 μM) was found
to be more effective than monastrol against OVCAR03 cancer cell line [12, 24, 25]. The presence of
benzyl ester at position 5 of DHPM [26] enhanced the anticancer activity as shown in compound 1,
(IC50 = 55 ± 3.37 μM) against MCF7 and HeLa (IC50 = 40 ± 2.43 μM) cancer cell lines [27], (Figure
1B). Moreover, replacement of ester with ketone analogues at 5 position resulted in a more potent
inhibitor, mon-97 (IC50 = 0.1 ± 0.02 μM) [23] (Figure 1C). In addition, cyclic ketone analogues
conferred rigidity to the conformation of DHPM, as presented in dimethylenastron (IC50 = 0.2 ± 0.11
μM), (Figure 1C) and improved the potency 100-time relative to monastrol [28]. Furthermore,
3

compound 2, the hybrid of DHPM and benzimidazole ring, (Figure 1D) exhibited anticancer activity
against PC3 (IC50 = 37 μM) and NCIH1299 (IC50 = 40 μM) cancer cell lines [29].
Motivated by the aforementioned findings, we have utilized rational chemical approaches to design
and synthesize novel DHPM derivatives as potential kinesin inhibitors for cancer therapy. In this
study, the integration of the benzyl ester moiety into the piperastrol skeleton afforded analogues 8a-
h which upon acylation of 3-NH of DHPM ring resulted in analogues 9a-d as shown in (Figure 1E).
However, incorporation of different ketone or cyclic ketone moieties into piperastrol skeleton yielded
analogues 10, 11 and 12 (Figure 1F). Moreover, we designed the new analogue, 15, (Figure 1G) as
a result of the amalgamation between active moieties; benzyl ester, benzimidazole with piperastrol
nucleus. Lastly, we designed the new analogue, 16, (Figure 1H) as a hybrid of ketone side chain,
heterocyclic benzimidazole and piperastrol.
Figure 1. The workflow of the rational structure-based design strategy in this study
4

3. Results and Discussion
3.1. Chemistry
As shown in Scheme 1, Williamson method was employed through the nucleophilic substitution
reaction between 3,4-dihydroxybenzaldehyde 3 and dichloromethane under basic condition to afford,
1,3-benzodioxole-5-carbaldehyde (piperonal) [30, 31].
O
OH
O
OH
i
O
O
4
3
Scheme 1. Synthesis of 1,3-benzodioxole-5-carbaldehyde 4. Reagents and conditions: (i) CH2Cl2, K2CO3, DMF, reflux,
12 h.
Different acetoacetate derivatives were prepared according to the modified Clemens method via
condensation of 2,2,6-trimethyl-1,3-dioxin-4-one (TMD) with different alcohols (5a-d) in refluxing
xylene [32-34] (Scheme 2).
O
O
O
i
O
+
R1-OH
R1
O
O
5a-d
TMD
6a-d
5a, 6a: R1= CH(CH3)2
5c, 6c: R1= CH2CH2OCH3
5b, 6b: R1= CH2CH(CH3)2 5d, 6d: R1= CH2C6H5
Scheme 2. Synthesis of acetoacetate derivatives 6a-d. Reagents and conditions: (i) TMD, xylene, reflux, 6 h.
As presented in Schemes 3 and 4, racemic mixture of DHPM derivatives, 8a-h, 10a-d, 11a-b and 12,
were conveniently prepared via multicomponent reaction, of Biginelli type [35]. Under acidic
condition, cyclocondensation of 1,3-benzodioxole-5-carbaldehyde 4, thiourea 7a or urea 7b and
active methylene compounds such as acetoacetates (6a-d) or β-diketones afforded racemic mixture
of target compounds 8a-h. 3-N-acetyl DHPM derivatives 9a-d in Scheme 3 were prepared by heating
DHPM derivatives (8a-d) with acetic anhydride for 5h [36]. In case of compound 12, it was
previously synthesized by microwave irradiation technique [37].
5

O
O
O
O
O
O
O
O
X
O
i
ii
R1
R1
O
NH
+
H2N
NH2
O
N
N
X
N
S
H
O
H
7a-b
8a-h
4
9a-d
8a: X=S, R1= CH(CH3)2
8b: X=S, R1= CH2CH(CH3)2
8c: X=S, R1= CH2CH2OCH3
8d: X=S, R1= CH2C6H5
9a: R1= CH(CH3)2
7a: X=S
7b: X=O
9b: R1= CH2CH(CH3)2
9c: R1= CH2CH2OCH3
9d: R1= CH2C6H5
8e: X=O, R1= CH(CH3)2
8f: X=O, R1= CH2CH(CH3)2
8g: X=O, R1= CH2CH2OCH3
8h: X=O, R1= CH2C6H5
Scheme 3. Synthesis of DHPM derivatives 8a-h and acetylated DHPM derivatives 9a-d. Reagents and conditions: (i)
Different acetoacetates (6a-d), citric acid, TEOF, reflux, 10 h; (ii) Acetic anhydride, reflux, 5 h.
O
O
O
O
O
O
O
X
O
ii
i
R2
NH
NH
+
H2N
NH2
N
X
N
X
H
O
H
7a-b
4
10a-d
11a-b
7a: X=S
7b: X=O
10a: X=S, R2=CH3
10b: X=S, R2=C6H5
10c: X=O, R2=CH3
10d: X=O, R2=C6H5
iii
11a: X=S
11b: X=O
O
O
O
NH
N
X
H
12
Scheme 4. Synthesis of monocyclic DHPM derivatives, 10a-d, tricyclic DHPM derivatives, 11a-b, and bicyclic DHPM
derivative, 12. Reagents and conditions: (i) Different β-diketones, citric acid, TEOF, reflux, 10 h; (ii) 1,3-
indandione, citric acid, TEOF, reflux, 10 h; (iii) Dimedone, citric acid, TEOF, reflux, 10 h.
2-Aminobenzimidazole 14 was synthesized by reacting 1,2-phenylenediamine 13 with cyanogen
bromide, followed by neutralization with concentrated ammonium hydroxide [38] as shown in
Scheme 5.
6

NH2
NH2
i
N
NH2
N
H
13
14
Scheme 5. Synthesis of 2-aminobenzimidazole 14. Reagents and conditions: (i) CNBr, ethanol/water, DMF, reflux, 1 h.
As reported in Scheme 6, one-pot cyclocondensation of 1,3-benzodioxole-5-carbaldehyde 4, 2-
aminobenzimidazole 14 with acetyl acetone or benzyl acetoacetate carried out in DMF afforded the
novel tricyclic DHPM derivatives, 15 and 16, respectively [29].
O
O
O
O
O
O
O
O
N
ii
i
O
N
NH2
+
N
N
H
N
N
N
N
H
O
H
15
14
16
4
Scheme 6. Synthesis of hybrid DHPM derivatives 15 and 16. Reagents and conditions: (i) Benzyl acetoacetate, DMF,
reflux, 10 h; (ii) Acetyl acetone, DMF, reflux, 10 h.
13
Elemental analysis and the spectroscopic data (1H NMR, C NMR, and mass spectroscopy) of the
synthesized compounds are in concord with the expected chemical structures as given in the
experimental section later.
The 1H NMR spectrum of compound 11b, as a representative example, was characterized by a singlet
signal at δ = 6.22 ppm of the two protons of dioxolane ring. A doublet signal at 7.14 ppm was
observed for the 6-H of the benzodioxole moiety. The 4-H proton of the dihydropyrimidine moiety
resonated at 7.78 ppm as a singlet. Besides, multiplet signals at 7.93 – 8.01 ppm with seven protons
integration, were assigned for; four protons at 6,7,8,9-H4 of indene moiety, one proton at 6-H of
benzodioxole moiety and two protons at 1,3-H2 of the dihydropyrimidine part. Moreover, a doublet
signal at 8.53 ppm is assigned for the 4-H of benzodioxole which showed meta-coupling to the 6-H
of benzodioxole moiety. Further, 1H-1H COSY spectrum of compound 11b supported this assignment
(See Supplementary Data).
13
13C NMR spectral assignment of all the synthesized compounds has been made. C NMR (on-
resonance & DEPT) spectra of compound 11b showed the signals resonated at: 102.94, 109.30,
112.32, 133.63, 139.75, and 148.23 ppm are assigned to seven carbons of benzodioxole moiety, while
the signals resonated at: 123.36, 123.47, 128.03, 136.15, 136.27, and 142.24 ppm are assigned to six
carbons of benzene ring of indene moiety. The three signals for the dihydropyrimidine core at 127.06
ppm for C-5, 146.42 ppm for C-4, and 152.79 ppm for C-6 were observed. The signals resonated in
7

1
the deshielded region at 189.53, and 190.20 ppm are assigned to two C=O carbons. Moreover, H-
13C Heteronuclear single quantum coherence (HSQC) spectrum of compound 11b supported this
assignment and added strong evidence for this elucidation (See supplementary material).
3.2.
Biological evaluation
3.2.1. In vitro anticancer screening at NCI-USA
The novel twenty-one synthesized compounds were acquiesced to the National Cancer Institute
"NCI" (www.dtp.nci.nih.gov), Bethesda, Maryland, USA. All submitted compounds were nominated
on the basis of the degree of structural diversity and computer modelling techniques for the evaluation
of their antineoplastic activity. The screening of activity passes through two possible processes,
commencing with the assessment of all compounds against 60 cell lines on nine types of human tumor
at a single dose of 10 μM. The output from the single dose screening is covered as a mean graph, and
it became available to be analyzed by the COMPARE program. Finally, the compounds which exhibit
significant growth inhibition get to pass the assessment against the 60 cell lines at five doses including
the one mentioned before.
3.2.1.1. In vitro single-dose (10 μM) anticancer screening on NCI 60 cell panels
The synthesized target compounds (8a-h, 9a-d, 10a-d, 11a-b, 12, 15 and 16) were initially subjected
to in vitro one dose anticancer assay in full NCI 60 cell lines panel represented by nine human
systems, including, Leukemia (L), Non-Small Cell Lung Cancer (NSCLC), Colon Cancer (CC),
Central Nervous System Cancer (CNSC), Melanoma (M), Ovarian Cancer (OC), Renal Cancer (RC),
Prostate Cancer (PC), and Breast Cancer (BC) in agreement with the protocol of the NCI, USA. The
compounds were added at 10 μM and the culture was incubated for 48h. Endpoint determinations
were carried out with a protein binding dye, sulforhodamine B. Later, the outcomes of all tested
compounds were described as a percentage growth of the treated cells compared to the unprocessed
control cells. The mean-graph midpoint values (MG-MID) are described in Table 1.
Table 1. The mean-graph midpoint (MG-MID) values of percentage growth of all tested compounds
Cpd.
no.
NSC
code
MG-
MID
L
NSCLC
CC
CNSC
M
OC
RC
PC
BC
808610
808612
800517
800516
808609
808611
809235
102.89
95.02
100.16
103.61
100.58
96.37
104.95
103.86
101.73
107.45
108.14
104.94
102.40
107.64
107.71
101.94
98.35
101.02
101.26
99.49
101.09
101.92
96.76
98.85
102.49
101.62
98.72
102.37
103.18
101.96
98.69
104.61
102.95
105.19
98.81
99.82
100.90
100.78
93.01
99.01
97.45
96.92
99.90
98.31
101.26
101.66
101.64
95.07
103.62
103.90
100.43
99.61
97.88
94.77
98.47
81.67
8a
8b
8c
8d
8e
8f
97.58
101.86
92.88
104.31
100.47
103.24
95.96
95.49
90.38
101.87
88.17
100.51
91.79
100.29
103.78
101.51
94.09
92.89
88.19
98.12
79.84
77.29
109.55
105.04
100.37
104.47
105.11
101.45
102.39
101.96
96.31
8g
8h
9a
9b
9c
9d
808618 100.73 100.59 105.83
97.42
97.61
96.61
95.30
80.12
97.53
810528
810529
810531
810530
87.12
81.34
98.27
50.65
94.22
93.53
95.41
87.32
101.34
98.97
101.81 103.66 102.09
98.96
99.64
79.46
97.35
98.98
94.70
98.58
97.97
89.76
103.16
73.71
8

809237
812017
809236
813491
809243
809242
100.63
25.88
102.41
100.21
104.17
104.49
103.27
54.06
102.04
99.60
107.04
32.33
106.20
103.93
106.60
108.00
98.38
43.89
100.58
98.40
96.35
95.22
104.37
38.25
104.52
99.15
104.68
50.76
105.90
101.89
104.74
108.00
99.64
45.01
100.96
97.70
99.05
99.23
104.45
70.10
108.33
104.00
104.30
107.52
104.22
49.55
103.12
99.53
102.94
43.81
103.41
100.17
102.51
103.25
10a
10b
10c
10d
11a
11b
12
100.91
101.95
105.86
104.72
100.99
102.66
808614 106.67 108.59 109.68 104.30 103.48 106.17 105.81 103.14 109.14 106.55
15
812015
810527
18.45
91.39
46.87
98.38
27.44
26.86
98.66
34.31
43.32
59.12
47.31
32.86
97.72
35.21
16
103.96
104.73 104.76 101.65 101.14
100.52
Compounds showed strong (10b and 15) and low (8d, 8h, 9a-d 12 and 16) cytotoxic activity selected for the next step,
one dose full panel analysis, are in bold.
Data analysis of the mean-graph midpoint (MG-MID) values revealed that, compounds 10b and 15
showed the most potent cytotoxicity with MG-MID values of 43.81% and 35.21% respectively.
Moreover, they showed broad spectrum activity against all tested cancer cell lines while, remain
compounds showed mild cytotoxic activity as indicated in Table 1.
Consequently, out of the screened twenty-one target compounds, ten hybrids composed of strong
(10b and 15) and low (8d, 8h, 9a-d 12 and 16) cytotoxic compounds were successfully taken to the
next step, one dose full panel analysis for evaluation of cytotoxic activity against NCI 60 human
tumor cell lines. The percentage of cell growth are presented in Table S1 (supplementary material).
Since the result reported for the single dose assay was growth percentage of the treated cells, this
allows detection of both growth inhibition (when subtract growth percentage from 100, values from
0 to 100) and lethality (values less than zero). For example, a growth percentage value of 100 means
zero growth inhibition. A growth percentage value of 60 would mean 40% growth inhibition. A
growth percentage value of zero means no net growth by the treatment. A growth percentage value
of -60 would mean 60% lethality. A value of -100 means all cells are dead [39].
As given in Table S1, compound 15 (NSC 812015) demonstrated the most remarkable and potent
cytotoxic activity against the different NCI cell panel. Regarding lethality, compound 15 displayed
lethal effect against leukemia, cancer HL-60 (TB) by (-14.74 %), CNS cancer SNB-75 by (-6.67 %),
melanoma MDA-MB-435 by (-35.36 %), and renal A498 cancer cell line (-10.15 %) at a single dose
assay concentration of 10-5 M. Furthermore, compound 10b revealed the lethal influence on CNS
cancer SNB-75 by (-1.40 %), melanoma MDA-MB-435 by (-49.49 %), and renal A498 cancer cell
line by (-12.48 %) at a single dose assay.
Data analysis of growth inhibition assay disclosed also that, compound 15 showed a discernible
potent activity against a wide variety of different cancer cell lines under investigation. As designated
in Table S1, Compound 15 displayed growth percentage less than 40 in all leukemia cell lines,
NSCLC; NCI-H460 and NCI-H522 cell lines, all colon cancer cell lines (except HCC-2998), all
CNSC cell lines (except SF-268 and SNB-19), Melanoma; LOX IMVI, MALME-3M, M14 and SK-
MEL-5 cell lines, ovarian cancer; OVCAR-3 and NCI/ADR-RES cell lines, renal cancer; ACHN and
9

CAKI-1 cell lines, breast cancer; MCF7, HS 578T and MDA-MB-468 cell lines. In addition,
compound 10b exhibited a potent activity against all leukemia cell lines (except CCRF-CEM and
RPMI-8226), NSCLC; NCI-H460 and NCI-H522 cell lines, all colon cancer cell lines (except COLO
205 and HCC-2998), CNSC; SF-539 and U251 cell lines, Melanoma; LOX IMVI, M14 and SK-
MEL-5 cell lines, ovarian cancer; OVCAR-3 cell line, renal cancer; CAKI-1 cell line, breast cancer;
MCF7 and HS 578T cell lines with growth percentage less than 40. Curiously, compound 9d showed
potent and selective cytotoxic activity against leukemia, cancer HL-60 (TB) cell line.
Compounds displayed growth percentage in the range of 60-80 are considered moderately active
cytotoxic agents. Data showed that, compound 15 showed moderate activity on the remaining of the
cancer cell lines. Moreover, Compound 10b showed moderate activity on leukemia; CCRF-CEM and
RPMI-8226 cell lines, NSCLC; A549, EKVX, HOP-92, NCI-H226, NCI-H23 and NCI-H322M,
colon cancer; COLO 205 and HCC-2998, cell lines, CNSC; SF-268, SF-295 and SNB-75, cell lines,
Melanoma; MALME-3M, SK-MEL-2, SK-MEL-28, UACC-257 and UACC-62, cell lines, ovarian
cancer; IGROV1, OVCAR-5, OVCAR-8, NCI/ADR-RES and SK-OV-3, cell line, all renal cancer
(except A498 and CAKI-1), all prostate cancer cell lines, all breast cancer (except MCF7 and HS
578T). Unfortunately, the activity of the other target compounds was in range of moderate (G%, 40-
80) and mild (G%, more than 80) depending on their growth percentage values.
3.2.1.1.1. Structure activity relationship
The rationale behind preparing a series of DHPM scaffold bearing different functionalities was to
establish a definite structure activity relationship pattern and emphasize the role of structure
manipulations in imparting bioactivity. Scanning Table 1, we have deduced that, the introduction of
sulfur atom into the 2-position of DHPM ring significantly influenced the antitumor activity. For
example, compounds 8d and 10b (MG-MID, 95.07 and 43.81 respectively) exhibited higher activity
than their oxo-analogues 8h and 10d (MG-MID, 99.61 and 100.17 respectively). On the other hand,
the introduction of acetyl group at the 3-position enhanced the anticancer activity as noticed in
compounds 9a-d with MG-MID values ranged from 97.80 to 81.67 when compared with their non-
acetylated analogues 8a-d with MG-MID values ranged from 101.60 to 95.07. Moreover, the
incorporation of a phenyl or benzyl group through a ketone or ester bridge to 5-position of the DHPM
ring resulted in significant enhancement of potency as reported in Table 1 for compounds 10b and
15 with MG-MID = 43.81 and 35.21 respectively. The presence of alkyl ester in compounds 8a-c
(MG-MID values ranged from 101.26 to 101.66) or alkyl ketone in analogues 10a, c, d (MG-MID
values ranged from 100.17 to 103.41) at 5-position of the DHPM ring revealed lower anticancer
activity. The inclusion of the carbonyl group at the 5-position into a cyclic structure either in a
cyclohexanone as observed in compound 12 (MG-MID = 106.55) or indenone ring in compounds
10

11a-b (MG-MID = 102.51 and 106.25 respectively) diminished the cytotoxic activity. Compound 15
(MG-MID = 35.22) having benzimidazole moiety and benzyl ester group has emerged as the most
promising antimitotic anticancer compound of this series while its methyl ketone analogue,
compound 16 demonstrated much lower potency with MG-MID value of 100.51. Accordingly,
substituents at different positions of the DHPM ring played an important role in controlling the
anticancer activity of our target compounds. Free nitrogen at position-1 as well as sulfur atom at
position-2 were favored, while free nitrogen at position-3 was not essential for activity. Finally,
lipophilic groups, such as phenyl and benzyl, at position-5 were markedly enhanced the activity. The
aforementioned SAR was supported by the docking study on later section.
3.2.1.2. In-vitro 5 doses full NCI 60 cell panel assay
The tested compounds which satisfied scheduled threshold growth inhibition criteria would further
be selected for NCI full panel five dose assay at tenfold dilutions of five concentrations (0.01, 0.1, 1,
10 & 100 μM) ranging from 10-4 to 10-8 M against all the NCI 60 tested cell lines. Accordingly,
compound 15 (NSC 812015), which fulfilled predetermined threshold inhibition criteria, was selected
for additional screening at 5-log dose molar range. The consequences, of cytotoxic activity of
compound 15, were used to create log concentration versus % growth inhibition curves and
accordingly, three response parameters (GI50, TGI & LC50) were computed for each cell line as listed
in Table S2. The GI50 value (growth inhibitory activity) relates to the concentration of the compound
initiating 50% decrease in the net cell growth while, the TGI value (cytostatic activity) is the
concentration of the compound resulting in total growth inhibition, and LC50 value (cytotoxic activity)
is the concentration of the certain compound causing net 50% loss of initial cells at termination of the
incubation period of 48 h [39].
It can be deduced from Table S2 that compound 15 (NSC 812015) exhibited remarkable anticancer
activity against most of the tested cell lines representing nine different subpanels at GI50 level
beginning from 0.08 μM. Analysis of data in Table S2 revealed that, compound 15 exhibited the
highest activity against MDA-MB-435 (Melanoma) with GI50 of 0.08 μM and least activity against
NCI-H226 (Non-Small Cell Lung Cancer), SK-MEL-28 (Melanoma), OVCAR-5 (ovarian cancer)
and TK-10 (Renal Cancer) with GI50 > 100 μM. The best value of TGI was noted on MDA-MB-435
(Melanoma) with 0.35 μM. Toxicity is measured in terms of lethality; compound 15 was not lethal and
safe in nature as it is obvious by examining the LC50 value >100 μM [40].
The criterion for selectivity of a compound depends upon its selective index, which was obtained by
dividing the full panel MIDa (the average sensitivity of all cell lines towards the test agent) by their
individual subpanel MIDb (the average sensitivity of all cell lines of a specific subpanel towards the
test agent). The ratios between 3 - 6 indicate moderate selectivity, ratios greater than 6 refer to high
11

selectivity to the corresponding cell line and compounds with ratios less than 3 will be non-selective
towards the corresponding subpanel. Compound 15 exhibited selectivity ratios ranging from 0.46 to
22.30 at the GI50 level. It displayed high selectivity towards CNS subpanel, prostate subpanel and
leukemia with selectivity ratios of 22.30, 15.38 and 12.56 at GI50 level respectively. It exhibited
moderate selectivity against colon subpanel with selectivity ratio of 4.65 at GI50 level, while it was
non-selectivity toward the NSCLC (selectivity ratio; 0.46), melanoma (selectivity ratio; 0.77),
ovarian cancer (selectivity ratio; 0.54), renal cancer (selectivity ratio; 0.61) and breast cancer
(selectivity ratio; 0.61) as shown in Table S2 and Figure 2 in comparison with monastrol.
25
20
15
10
5
15
0
Monastrol
SUBPANEL TUMOR CELL LINES
Figure 2. Subpanel tumor cell lines selectivity ratios of compound 15 and monastrol at GI50 level
The mean-graph midpoint (MG-MID) GI50 values comparison between compound 15 and monastrol
as a standard are given in Table 2. It can be remarked that compound 15 displayed, comparatively
superior activity over monastrol where, the data indicated that the MID for compound 15, (8.92 μM)
was a lot less than that of the MID (50.10 μM) calculated for monastrol (Table 2).
Table 2. Comparison of compound 15 with monastrol at MG-MID GI50 level at the five doses
Compound
15
L
NSCLC
19.21
CC
1.92
41.94
CNSC
0.4
M
OC
RC
PC
BC
MG-MID
8.92
0.71
11.64 16.52 14.67 0.58 14.67
48.57 66.99 62.76 59.6 43.24
Monastrol 30.52
48.03
49.27
50.10
Dose response curves (percentage growth verses concentration of compound at NCI protocol, μM)
compound 15 against entirely cell lines in the NCI 60 panel are given in Figures 3 and S69.
12

Colors-codes of different cancer cell
lines: red, leukemia cell line; blue, lung
cancer; green, colon cancer; gray,
central nervous system cancer; coral,
melanoma; purple, ovarian cancer;
gold, renal cancer; turquoise, prostate
cancer; pink, breast cancer.
Figure 3. Collective dose response curves for entirely cell lines in the NCI 60 panel exposed to compound 15 (NSC
812015) with tissue originated colors.
3.2.2. Kinesin enzyme inhibition assay
Our synthesized target compounds were designed as inhibitors of kinesin enzyme which would lead
to cell cycle arrest at mitosis and ultimately, to cell death. Consequently, the kinesin enzymatic assays
for the most active DHPM derivatives were achieved, using monastrol and the potent inhibitor, mon-
97 as references. The five selected compounds 9d, 10b, 12, 15 and 16 exhibited higher inhibitory
activity, in micro molar range (IC50 values from 3.86 to 14.07 μM), in comparison to the positive
control, monastrol, which reported (IC50 = 20 μM), as indicated in Table 3. Consequently, we can
conclude that the introduction of sulfur atom at the 2-position and the 1,3-benzodioxole moiety at
position 4 were contributed to enhance the inhibitory activity of the DHPM scaffold as observed for
all five tested compounds. The presence of phenyl group at the side chain at 5-position remarkably,
initiated the inhibitory activity of compounds, 9d, 10b and 15. In addition, benzimidazole analogues
reported superior activity (compound 15). Interestingly, the dimethyl cyclohexyl hydrophobic moiety
lead to the most potent inhibitor, compound 12, with IC50 = 3.95 μM surpassing that of all other tested
compounds as well as the positive controls, monastrol and mon-97. Compound 9d displayed ‘cell
drop-off’ (i.e., lower potency in the cellular assay than in the enzyme assay) while it was the most
potent inhibitor of kinesin with IC50 of 3.86 ± 0.12 μM (Table 3), it displayed poor biochemical
efficacy towards mitotic kinesin target with MG-MID = 81.67 (Table 1). Science most known drug
targets and metabolizing enzymes are located inside cells, compound 9d could be metabolized rapidly
or bind to other proteins outside or inside the cell cultures leading to its poor intracellular
bioavailability and activity [41].
13

Table 3. Kinesin enzyme inhibitory activity for compounds 9d, 10b, 12, 15 and 16.
a
Compound
IC50
±
SDb (μM)
3.86 0.12
9d
10b
±
10.70 0.35
±
3.95 0.12
12
±
4.36 0.14
15
±
14.07 0.45
16
±
20 0.42
Monastrol
Mon-97
±
0.1 0.02
±
aThe half maximal inhibitory concentration, bthe standard deviation of triplicate experiments
3.2.3. In vitro cytotoxicity against human normal cell (Human Embryonic Kidney)
To investigate whether the synthesized compounds showed selective cytotoxicity between normal
and cancer cells, the most active target compounds 9d, 10b, 12, 15 and 16 were evaluated against
human embryonic kidney (HEK 293); a human normal cell line. As displayed in Table 4, all tested
compounds 9d, 10b, 12, 15 and 16 exhibited lower toxicity towards normal cell, HEK 293, with IC50
= 27.99, 62.02, 33.20, 16.75 and 19.30 μM/ml respectively in comparison to doxorubicin as a
reference drug (IC50 = 11.34 μM/ml). On the basis of this preceding information, the safest compound
among test compounds on HEK 293 is 10b about 5 times than doxorubicin. The representative IC50
values and curves showed in the SI in Table S3 and Figures S70-S75.
Table 4. Cytotoxic activity (IC50) of 9d, 10b, 12, 15 and 16 against human embryonic kidney normal cell (HEK 293)
a
Compound
IC50
±
SDb (μM/ml)
27.99 1.09
9d
±
62.02 2.42
10b
±
33.20 1.29
12
±
16.75 0.65
15
16
±
19.30 0.76
±
11.34 0.44
Doxorubicin (DOX)
±
aThe half maximal inhibitory concentration, bthe standard deviation of triplicate experiments
3.2.4. Cell cycle analysis
Targeting the cancer cell cycle has been emerged as a promising approach for cancer therapy [42].
To recognize the role of compound 15 in growth inhibition of cancer cells, and consequently,
induction of apoptosis in different phases, DNA flow cytometric analysis was used to measure the
effect of compound 15 and monastrol on induction of cell cycle of SNB-75 cancer cells. SNB-75 cells
were treated with compound 15 and monastrol at IC50 concentration (2 μM) for 24 h, stained with
propidium iodide (PI), analyzed by flow cytometer and the results were reported in Table 5.
A significant increase in the percentage of apoptotic cells at the pre-G1 phase, (24.82% on exposure
to 15 and 22.58% for monastrol) compared to control (1.85%), was detected with concurrent
reduction in the G0/G1 phase, 34.52% for 15 & 29.56% for monastrol, and compared to the control
(55.62%). Furthermore, a significant increase in the cells at G2/M phase, 37.59% for 15 & 41.26%
14

for monastrol, was detected compared to control (10.46%) as shown in Figure 4A. Both arrest of
G0/G1 phase and alteration of the pre-G1 phase are considered significant remarks for compound 15
to induce apoptosis in SNB-75 cells.
Table 5. Effect of compound 15 and monastrol on the cell cycle phases in SNB-75 cells.
Cell cycle distribution (%)
Compound
G0-G1
34.52
29.56
55.62
S
G2-M
37.59
41.26
10.46
pre-G1
24.82
22.58
1.85
15
27.89
29.18
33.92
Monastrol
Control
3.2.5. Detection of apoptosis
Extrinsic as well as intrinsic apoptosis in SNB-75 cells induced by compound 15 were evaluated by
annexin V and PI staining. Herein, SNB-75 cells were incubated with compound 15 and monastrol at
2 μM concentration for 24 h. Compound 15 induced an early apoptosis, (7.65%), in SNB-75 at 24 h
compared to monastrol (4.72%) and enhanced late apoptotic induction, (15.12%) by more than 25-
fold over the untreated cells (0.59). Thus, compound 15 induced apoptosis with 22.77%, and the
results were compared with monastrol (20.26%) (Figure 4B). This determination was consistent with
the data obtained from cell cycle analysis in Figure 4A.
A
15
Monastrol
Control
B
15
Monastrol
Control
Figure 4. A) Effect of compound 15 on the cell cycle distribution of SNB-75 cell line. B) Apoptosis effect on SNB-75
cell line induced by compound 15, the four quadrants identified as: LL: viable; LR: early apoptotic; UR: late
apoptotic; UL: necrotic.
15

3.2.6. Effects on the levels of active caspase-3 and caspase-9
Caspases, cysteine-containing aspartic acid-specific proteases, provide essential links in cell
controlling networks regulatory cell death. Caspase-3 is a crucial executioner protease which is
triggered by upstream initiator caspases as caspase-9 [43, 44].
This study was further extended to investigate the ability of compounds 9d and 15 to provoke
apoptosis in MDA-MB-435 melanoma cell line. Treatment of MDA-MB-435 cell line with
compound 9d significantly increased the expression levels of active caspases 3 and 9 by about 7 and
18 folds, respectively, while in case of compound 15 the levels of active caspases-3 and caspases-9
increased considerably by about 6 and 12 folds, respectively, in comparison to the control.
Interestingly, compound 9d displayed superior effect on both caspase-3 and caspases-9 over
monastrol as indicated in Table 6.
Table 6. Effect of compounds 9d and 15 on the active caspase-3 and caspase-9 level in MDA-MB-435 cells.
Caspase-3
Caspase-9
Compound
Pg/ml
354.20
306.20
309.20
44.80
Fold
7.90
6.83
6.90
1
ng/ml
22.12
14.63
14.03
1.18
Fold
18.79
12.43
11.92
1
9d
15
Monastrol
Control
3.3. Molecular docking
We have designed and synthesized novel series of DHPM as inhibitors of mitotic kinesin Eg5. In
order to understand the protein-ligand interaction, we have performed molecular docking studies
using AutoDock Vina [45] and UCSF Chimera (version 1.13.1) programs.
Kinesin is composed of an N-terminal motor domain bound through a linker to a central coiled-coil
and a C-terminal tail. The motor domain interacts with microtubules, and it is also responsible for
ATP hydrolysis and force generation [46, 47]. The allosteric binding site of Eg5 has been studied
through molecular modeling techniques and has been found to contain three hydrophobic sub pockets
(P1–P3) which interact with the inhibitors [48-51].
some demonstrative compounds, 9d, 10b, 12, 15, 16 and monastrol were docked into the crystal
structure of Eg5 motor domain, (PDB ID code 1Q0B) [48] allosteric binding site and the best poses
were evaluated by docking energy score values and their alignment with co-crystalized monastrol
were shown in Figures S76-81. Docking of R- and S-enantiomers of selected compounds revealed
that S-enantiomers exhibited better interaction and affinity than R- enantiomers as shown in Table
S4. Analysis of the molecular docking results showed that the interactions within the active site were
attributed to hydrogen bonds, hydrophobic and electrostatic attraction forces. Compounds 9d, 10b,
12, 15, and 16 were fitted well into the allosteric binding site. The resulted molecular docking files
were analyzed using Discovery Studio (DS) visualizer program [52] and the docking results were
16

reported in Table 7.
Table 7. Analysis of binding interaction of S-enantiomers of 9d, 10b, 12, 15, 16 and monastrol with KSP allosteric
binding site
Compound
code
Hydrogen bonding
interaction
Electrostatic
interaction
IC50 (μM)
Hydrophobic interaction
Thr112, Glu118, Arg119, Ile136, Leu160,
Tyr211, Leu214, Glu215, Gly217, Ala218,
Arg221, Phe239
Glu118, Arg119, Ile136, Pro137, Leu160,
Tyr211, Leu214, Gly217, Ala218, Arg221,
Phe239
Thr112, Glu118, Arg119, Ile136, Pro137,
Leu160, Tyr211, Leu214, Glu215, Gly217,
Ala218, Phe239
Thr112, Gly117, Glu118, Arg119, Ile136,
Pro137, Leu160, Tyr211, Leu214, Glu215,
Ala218, Arg221, Phe239
Glu116,
Arg221
9d
10b
12
3.86
Gly117, Arg221
Glu116, Gly117
Glu116, Gly117
Tyr211
10.70
3.95
4.36
Glu116
Glu116,
Arg221
15
Glu116
Thr112, Glu118, Arg119, Ile136, Pro137,
Leu214, Ala218, Arg221
Gly117, Arg119, Ile136, Tyr211, Leu214,
Glu215, Ala218
16
14.07
20
Gly117
Glu116
none
Monastrol
Glu116, Glu118
The most active analogue, 9d, forms two H-bonds, one with the back bone carbonyl oxygen of Gly117
in the allosteric binding pocket. The second H-bond is formed with the proton of the NH2 of Arg221
located in a minor cooperative binding pocket [53]. Moreover, it develops electrostatic attraction
forces, aromatic π-anion and π-cation interactions, with two amino acid residues, Glu116 and Arg221
respectively. Besides, strong hydrophobic interactions between aromatic rings with various
hydrophobic amino acid residues have been confirmed as shown in Figure 5. There was a minor
cooperative binding pocket surrounded by Ala218 and Arg221.
Figure 5. The 2D (right) and 3D (left) of the best docked poses showing interactions of S-enantiomer of 9d with the
amino acids of the active site of KSP.
The observed lower biological activity of analogue, 10b, could be explained by forming only one H-
bond with either the carbonyl group of the nearby amino acid residues, Glu116 or Gly117. In addition,
17

it has only one electrostatic aromatic π-anion interaction with the acidic amino acid Glu116 as
presented in Figure 6.
Figure 6. The 2D (right) and 3D (left) of the best docked poses showing interactions of S-enantiomer of 10b with the
amino acids of the active site of KSP.
The high potency of compound 12, could be explained by the additional hydrophobic π-alkyl
attraction forces between the two methyl groups located in the cyclohexanone ring with Ala218 and
Arg119. Furthermore, the electrostatic aromatic π-anion and π-cation interactions with the amino acid
residues, Glu116 and Arg221 were established (Figure 7).
Figure 7. The 2D (right) and 3D (left) of the best docked poses showing interactions of S-enantiomer of 12 with the
amino acids of the active site of KSP.
The relatively high activity of compound 15 is elucidated by the unique H-bond formed between the
benzimidazole-nitrogen and the proton of the hydroxyl group of Tyr211 in addition to the
18

hydrophobic attraction forces between benzyl ester with Ile136 and Leu214. It forms hydrophobic
aromatic π-anion interaction with Glu116. Furthermore, a face to face aromatic π-π- stacking of
benzimidazole ring with the benzene ring of Tyr211 is confirmed as shown in Figure 8.
Figure 8. The 2D (right) and 3D (left) of the best docked poses showing interactions of S-enantiomer of 15 with the
amino acids of the active site of KSP.
Compound 16, has relatively low potency compared to analogue 15 because of the absence of the
benzyl ester side chain and consequently the hydrophobic interaction with the corresponding amino
acid residues in the active site. Moreover, it lost the π-π stacking as well as the H-bond between
benzimidazole ring and Tyr211. It forms only one H-bond with Gly117 as well as electrostatic
aromatic π-anion interaction with Glu116. Weaker hydrophobic interactions with hydrophobic nearby
amino acid were also informed (Figure 9)
Figure 9. The 2D (right) and 3D (left) of the best docked poses showing interactions of S-enantiomer of 16 with the
amino acids of the active site of KSP
19

Besides the observed attraction forces of the selected compounds with the main binding pocket, there
was a minor cooperative binding pocket surrounded by Ala218 and Arg221. Binding to the latter
pocket enhanced the binding affinity of the studded compounds compared to those reported for
monastrol (Table 7).
4. Conclusion
In this study, novel series of twenty-one DHPM derivatives were designed and synthesized according
to Biginelli method. The anticancer activity of all of these derivatives, were tested at a single dose of
10-5 M concentration at the NCI (USA) over 60 cell line panel, and DHPM hybrid with benzimidazole
moiety 15 was subsequently tested in 5-doses testing mode. Hybrid 15 was identified as the most
promising anticancer candidate against most of cell lines. The inhibitory activity against kinesin
enzyme was also investigated for selected compounds 9d, 10b, 12, 15 and 16 with IC50 ranges, 3.86
- 14.07 μM (exceed monastrol). Compound 9d exhibited the best inhibitory activity with IC50 value
at 3.86 μM. The safest compound among test compounds on normal cell line (HEK 293) is 10b (IC50
= 62.02 ± 2.42 µM/ml) about 5 times than doxorubicin. Flow cytometric analysis indicated that the
SNB-75 cells were arrested in G2/M phase by compound 15. The effects of compounds 9d and 15 on
the active caspase-3 and caspase-9 levels were also investigated. The most active kinesin inhibitors,
9d, 10b, 12, 15 and 16 were docked into the kinesin Eg5 binding site, which developed significant
attraction forces with various amino acid residues in the active site better than monastrol. The DHPM
analogues discovered in this study may provide valuable therapeutic intervention for cancer
treatment.
5. Experimental
5.1. Chemistry
5.1.1. General
All organic reagents used were obtained from Sigma-Aldrich company, Alfa Aesar company or
Merck company and were used without further purification. All potential Eg5 inhibitors were
synthesized in their racemic form (±) using the well-known Biginelli three component reaction. The
optical rotations (α) of all synthesized compounds were recorded on a Polax-2L Polarimeter (ATAGO
Co., Ltd., Japan) at the department of pharmaceutical chemistry, Faculty of Pharmacy, Tanta
University, Egypt and they didn’t show a rotation. Melting points were determined on an electro-
thermal melting point apparatus (Stuart SMP10) by the open capillary method and were reported
uncorrected. Reactions were monitored by TLC using pre-coated sheet (Fastman Kodak Co., Silica
60 F254) using developing systems: n-hexane: ethyl acetate (40: 60) and were visualized with UV light
at 254 nm. Elemental analysis (% C, H, N, and S) was carried out by Perkin–Elmer 2400 CHNS
analyzer and were within ± 0.40 of the theoretical values. Elemental analysis was performed by the
20

regional center for mycology and biotechnology, Al-Azhar University, Nasr City, Cairo, Egypt. 1H,
13C NMR, DEPT-135, 2D COSY and HSQC spectra were recorded on Bruker FT-NMR spectrometer
at (400 MHz) and (100 MHz) respectively using DMSO-d6 as a solvent. A mixture of DMSO-d6 and
DMF was used to dissolve compounds 10b and 10d. All chemical shift values, coupling constants, J,
and multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet, br = broad, dd = doublet of doublet)
were quoted in ppm and in Hz respectively. 1H and 13C NMR spectra were performed by Faculty of
Pharmacy, Mansoura University, Egypt. Electron ionization mass spectra (EI-MS) were recorded on
Thermo Scientific, ISQ Single Quadruple MS, with ionization energy of 70 eV, and Helium gas was
used as the carrier gas at a constant flow rate of 1ml/min. Mass spectroscopy was performed by the
regional center for mycology and biotechnology, Al-Azhar University, Nasr City, Cairo, Egypt.
5.1.2. 1,3-Benzodioxole-5-carbaldehyde (Piperonal) 4
A solution of 3,4-dihydroxybenzaldehyde 3 (5.0 g, 36 mmol) in DMF (25 ml) was added dropwise
to a suspension of dichloromethane (3.5 ml, 54 mmol) and potassium carbonate (10 g, 72 mmol) in
DMF (25 ml). The mixture was heated under reflux for 12 h then cooled and filtered. The filtrate was
evaporated under vacuum. The remaining residue was dissolved in water and then extracted with
diethyl ether (3 x 30 ml). The ethereal extracts were collected, washed with sodium hydroxide (30
ml, 10 %) followed by distilled water (30 ml), dried over anhydrous magnesium sulfate and finally
the ether layer was evaporated under vacuum. The formed precipitate was crystallized from EtOH to
o
o
give compound 4 (3.5 g, 65%) as a light yellow solid with m.p. 37-39 C (Lit. m.p. 35-39 C) [30,
31].
5.1.3. General procedure for preparation of acetoacetic esters derivatives (6a-d)
A solution of the appropriate alcohol, 5a-d, (25 mmol) and TMD (3.5 g, 25 mmol) in 10 ml xylene
was heated under reflux in an oil bath at 150 oC, for 6 h. The reaction mixture was cooled and then
xylene was removed under reduced pressure to yield products, 6a-d which were in a high level of
purity and were used immediately in subsequent reactions [34]. 6a, a yellow oil, with bp 183-185 oC,
(Lit. bp 185-187 oC), 6b, a yellow oil, with bp 200-201 oC, (Lit. bp 204-205 oC), 6c, a yellow oil,
with bp 115-117 oC, (Lit bp 120-122 oC) and 6d, a yellow oil, with bp 270-271 oC, (Lit bp 275-276
oC) [54].
5.1.4. General procedure for preparation of DHPM compounds (8a–h)
A mixture of acetoacetate esters 6a-d (25 mmol), compound 4 (25 mmol), thiourea 7a / urea 7b (50
mmol), citric acid (25 mmol) and triethylorthoformate (50 mmol) was heated under reflux for 10 h.
The reaction was quenched by addition of water (15 ml) and then cooled in an ice-bath. The
precipitated solid was filtered, and recrystallized from isopropanol to afford products (8a-h).
5.1.4.1. (±)-isopropyl 4-(benzo[d][1,3]dioxol-5-yl)-6-methyl-2-thioxo-1,2,3,4-tetrahydro-
21

pyrimidine-5- carboxylate (8a)
Yield (0.23 g, 66 %) as white powder with m.p. 175-176 oC. 1H NMR (400 MHz, DMSO-d6) δ (ppm):
1.03 (3H, d, J = 6.0 Hz, CHCH3), 1.18 (3H, d, J = 6.0 Hz, CHCH3), 2.29 (3H, s, CH3), 4.84 – 4.87
(1H, m, COOCH), 5.08 (1H, d, J = 1.6 Hz, pyrimidine 4-H), 6.01 (2H, s, OCH2O), 6.68 (1H, dd, J =
8.0, 1.6 Hz, 6-H), 6.73 (1H, d, J = 1.6 Hz, 4-H), 6.89 (1H, d, J = 8.0 Hz, 7-H), 9.59 (1H, d, J = 1.6
Hz, 3-NH) , 10.29 (1H, s, 1-NH). 13C NMR (100 MHz, DMSO) δ (ppm): 17.57, 21.93, 22.18, 54.24,
67.32, 101.46, 101.54, 107.20, 108.57, 120.17, 138.05, 145.25, 147.13, 147.81, 165.06, 174.45. EI-
MS: m/z: 334.02 [M+]. Anal. Calcd. for C16H18N2O4S: C, 57.47; H, 5.44; N, 8.38; S, 9.59. Found: C,
57.67; H, 5.56; N, 8.68; S, 9.77.
5.1.4.2. (±)-isobutyl 4-(benzo[d][1,3]dioxol-5-yl)-6-methyl-2-thioxo-1,2,3,4-tetrahydro-
pyrimidine-5-carboxylate (8b)
Yield (0.19 g, 53 %) as faint yellow powder with m.p. 134 -135 oC. 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 0.77-0.78 (6H, m, CH(CH3)2), 1.75 – 1.81 (1H, m, CH(CH3)2), 2.33 (3H, s, CH3), 3.74 (1H,
dd, J = 10.8, 6.4 Hz, COOCH2), 3.81 (1H, dd, J = 10.8, 6.4 Hz, COOCH2), 5.11 (1H, d, J = 2.8 Hz,
pyrimidine 4-H), 5.99 (2H, s, OCH2O), 6.69 (1H, dd, J = 8.0, 1.6 Hz, 6-H), 6.74 (1H, d, J = 1.6 Hz,
4-H), 6.88 (1H, d, J = 8.0 Hz, 7-H), 9.59 (1H, d, J = 2.8 Hz, 3-NH) , 10.33 (1H, s, 1-NH). 13C NMR
(100 MHz, DMSO) δ (ppm): 17.67, 19.27, 19.33, 27.68, 54.17, 70.04, 100.81, 101.55, 107.23,
108.66, 120.17, 137.72, 145.94, 147.20, 147.90, 165.57, 174.33. EI-MS: m/z: 348.01 [M+]. Anal.
Calcd. for C17H20N2O4S: C, 58.60; H, 5.80; N, 8.04; S, 9.20. Found: C, 58.60; H, 5.52; N, 7.98; S,
8.99.
5.1.4.3. (±)-2-methoxyethyl 4-(benzo[d][1,3]dioxol-5-yl)-6-methyl-2-thioxo-1,2,3,4-tetrahydro-
pyrimidine-5-carboxylate (8c)
Yield (0.25 g, 69 %) as faint yellow powder with m.p. 179 -181 oC. 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 2.29 (3H, s, CH3), 3.22 (3H, s, OCH3), 3.45-3.50 (2H, m, CH2OCH3), 4.05-4.17 (2H, m,
COOCH2), 5.11 (1H, d, J = 2.8 Hz, pyrimidine 4-H), 6.00 (2H, s, OCH2O), 6.70 (1H, dd, J = 8.0, 1.6
Hz, 6-H), 6.75 (1H, d, J = 1.6 Hz, 4-H), 6.88 (1H, d, J = 8.0 Hz, 7-H), 9.62 (1H, d, J = 2.8 Hz, 3-
NH), 10.36 (1H, s, 1-NH). 13C NMR (100 MHz, DMSO) δ (ppm): 17.61, 54.06, 58.43, 63.14, 70.24,
101.13, 101.54, 107.20, 108.64, 120.02, 137.95, 145.75, 147.15, 147.84, 165.48, 174.65. EI-MS: m/z:
349.96 [M+]. Anal. Calcd. for C16H18N2O5S: C, 54.84; H, 5.19; N, 8.00; S, 9.15. Found: C, 54.97; H,
5.45; N, 8.20; S, 9.43.
5.1.4.4. (±)-benzyl 4-(benzo[d][1,3]dioxol-5-yl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-
5-carboxylate (8d)
Yield (0.28 g, 71 %) as yellow powder with m.p. 202-204 oC, (Lit. m.p. 204 oC) [15]. 1H NMR (400
MHz, DMSO-d6) δ (ppm): 2.33 (3H, s, CH3), 5.03 (1H, d, J = 12.4 Hz, COOCH2), 5.12 (1H, d, J =
12.4 Hz, COOCH2), 5.14 (1H, d, J = 1.6 Hz, pyrimidine 4-H), 6.01 (2H, s, OCH2O), 6.66 (1H, dd, J
22

= 8.0, 1.6 Hz, 6-H), 6.71 (1H, d, J = 1.6 Hz, 4-H), 6.86 (1H, d, J = 8.0 Hz, 7-H), 7.17 – 7.33 (5H, m,
Bn-H5), 9.62 (1H, d, J = 1.6 Hz, 3-NH), 10.37 (1H, s, 1-NH). 13C NMR (100 MHz, DMSO) δ (ppm):
17.71, 54.18, 65.59, 100.63, 101.58, 107.33, 108.64, 120.24, 128.06, 128.29, 128.77, 136.74, 137.77,
146.30, 147.24, 147.89, 165.32, 174.36. EI-MS: m/z: 382.02 [M+]. Anal. Calcd. for C20H18N2O4S:
C, 62.81; H, 4.75; N, 7.33; S, 8.39. Found: C, 62.56; H, 4.54; N, 7.09; S, 8.55.
5.1.4.5. (±)-isopropyl 4-(benzo[d][1,3]dioxol-5-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-
5-carboxylate (8e)
Yield (0.28 g, 85 %) as white powder with m.p. 192-193 oC, (Lit. m.p. 194-196 oC) [55]. 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 1.02 (3H, d, J = 6.4 Hz, CHCH3), 1.17 (3H, d, J = 6.4 Hz, CHCH3),
2.25 (3H, s, CH3), 4.80 – 4.85 (1H, m, COOCH), 5.07 (1H, s, pyrimidine 4-H), 5.99 (2H, s, OCH2O),
6.70 (1H, d, J = 8.0 Hz, 6-H), 6.75 (1H, s, 4-H), 6.86 (1H, d, J = 8.0 Hz, 7-H), 7.67 (1H, s, 3-NH) ,
9.15 (1H, s, 1-NH). 13C NMR (100 MHz, DMSO) δ (ppm): 18.18, 21.98, 22.25, 54.21, 66.79, 100.02,
101.39, 107.14, 108.41, 119.86, 139.46, 146.77, 147.67, 148.51, 152.51, 165.28. EI-MS: m/z: 318.16
[M+]. Anal. Calcd. for C16H18N2O5: C, 60.37; H, 5.71; N, 8.80. Found: C, 60.03; H, 5.72; N, 8.65.
5.1.4.6. (±)-isobutyl 4-(benzo[d][1,3]dioxol-5-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (8f)
Yield (0.24 g, 70 %) as white powder with m.p. 181-183 oC. 1H NMR (400 MHz, DMSO-d6) δ (ppm):
0.78 (6H, d, J = 5.2 Hz, CH(CH3)2), 1.75 – 1.80 (1H, m, CH(CH3)2), 2.29 (3H, s, CH3), 3.72 (1H, d,
J = 6.2 Hz, COOCH2), 3.77 (1H, d, J = 6.2 Hz, COOCH2), 5.09 (1H, s, pyrimidine 4-H), 5.99 (2H, s,
OCH2O), 6.70 (1H, d, J = 7.6 Hz, 6-H), 6.75 (1H, s, 4-H), 6.86 (1H, d, J = 7.6 Hz, 7-H), 7.68 (1H, s,
3-NH) , 9.19 (1H, s, 1-NH). 13C NMR (100 MHz, DMSO) δ (ppm): 18.27, 19.33, 19.37, 27.72, 54.11,
69.74, 99.39, 101.40, 107.13, 108.52, 119.80, 139.16, 146.83, 147.75, 149.24, 152.43, 165.79. EI-
MS: m/z: 332.04 [M+]. Anal. Calcd. for C17H20N2O5: C, 61.43; H, 6.08; N, 8.43. Found: C, 61.21; H,
6.31; N, 8.52.
5.1.4.7. (±)-2-methoxyethyl 4-(benzo[d][1,3]dioxol-5-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydro-
pyrimidine -5-carboxylate (8g)
o
1
Yield (0.30 g, 86 %) as white powder with m.p. 146 -148 C. H NMR (400 MHz, DMSO-d6) δ
(ppm): 2.25 (3H, s, CH3), 3.23 (3H, s, OCH3), 3.44 – 3.49 (2H, m, 8.8 Hz, CH2OCH3), 4.06 – 4.10
(2H, m, COOCH2), 5.09 (1H, d, J = 3.2 Hz, pyrimidine 4-H), 5.98 (2H, s, OCH2O), 6.72 (1H, dd, J
= 8.0, 1.4 Hz, 6-H), 6.77 (1H, d, J = 1.4 Hz, 4-H), 6.85 (1H, d, J = 8.0 Hz, 7-H), 7.70 (1H, d, J =
3.2 Hz, 3-NH), 9.21 (1H, s, 1-NH). 13C NMR (100 MHz, DMSO) δ (ppm): 18.23, 53.98, 58.43, 62.81,
70.34, 99.69, 101.39, 107.15, 108.49, 119.71, 139.33, 146.80, 147.70, 149.10, 152.62, 165.68. EI-
MS: m/z: 334.00 [M+]. Anal. Calcd. for C16H18N2O6: C, 57.48; H, 5.44; N, 8.38. Found: C, 57.76; H,
5.61; N, 8.22.
23

5.1.4.8. (±)-benzyl 4-(benzo[d][1,3]dioxol-5-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (8h)
Yield (0.30 g, 79 %) as white powder with m.p. 166-168 oC. 1H NMR (400 MHz, DMSO-d6) δ (ppm):
2.28 (3H, s, CH3), 5.01 (1H, d, J = 12.8 Hz, COOCH2), 5.09 (1H, d, J = 12.8 Hz, COOCH2), 5.12
(1H, d, J = 2.6 Hz, pyrimidine 4-H), 5.99 (2H, s, OCH2O), 6.68 (1H, dd, J = 8.0, 1.4 Hz, 6-H), 6.72
(1H, d, J = 1.4 Hz, 4-H), 6.83 (1H, d, J = 8.0 Hz, 7-H), 7.17 – 7.33 (5H, m, Bn-H5), 7.70 (1H, d, J =
2.6 Hz, 3-NH), 9.25 (1H, s, 1-NH). 13C NMR (100 MHz, DMSO) δ (ppm): 18.33, 65.24, 66.45, 99.17,
101.43, 107.24, 108.51, 119.89, 127.98, 128.19, 128.49, 128.74, 128.90, 137.01, 139.17, 146.88,
147.76, 149.73, 152.35, 165.51. EI-MS: m/z: 366.02 [M+]. Anal. Calcd. for C20H18N2O5: C, 65.57;
H, 4.96; N, 7.65. Found: C, 65.24; H, 4.81; N, 7.49.
5.1.5. General procedure for preparation of acetylated DHPM compounds (9a–d)
A solution of 8a-d (5 mmol) in 15 ml of acetic anhydride was heated under reflux for 5 h. The solution
was then poured into 150 ml of ice-water and stirred for 12 hours until complete precipitation of the
product. The precipitate was filtered and then recrystallized from acetone to yield compounds (9a-
d).
5.1.5.1. (±)-Isopropyl 3-acetyl-4-(benzo[d][1,3]dioxol-5-yl)-6-methyl-2-thioxo-1,2,3,4-tetrahydro-
pyrimidine -5-carboxylate (9a)
o
1
Yield (0.22 g, 44 %) as yellow powder with m.p. 165-166 C. H NMR (400 MHz, DMSO-d6) δ
(ppm): 1.16 (3H, d, J = 6.0 Hz, CHCH3), 1.22 (3H, d, J = 6.0 Hz, CHCH3), 2.35 (3H, s, COCH3),
2.63 (3H, s, CH3), 4.95 – 4.98 (1H, m, COOCH), 6.00 (2H, s, OCH2O), 6.31 (1H, s, pyrimidine 4-
H), 6.66 (2H, m, 4,6-H2), 6.87 (1H, d, J = 7.6 Hz, 7-H), 11.67 (1H, s, 1-NH). 13C NMR (100 MHz,
DMSO) δ (ppm): 16.93, 22.06, 22.12, 27.67, 53.01, 68.26, 101.67, 107.01, 108.07, 108.63, 120.13,
133.27, 145.11, 147.34, 147.89, 164.76, 173.55, 178.28. EI-MS: m/z: 376.17 [M+]. Anal. Calcd. for
C18H20N2O5S: C, 57.43; H, 5.37; N, 7.44; S, 8.52. Found: C, 57.01; H, 5.34; N, 7.19; S, 8.31.
5.1.5.2. (±)-Isobutyl 3-acetyl-4-(benzo[d][1,3]dioxol-5-yl)-6-methyl-2-thioxo-1,2,3,4-tetrahydro-
pyrimidine -5-carboxylate (9b)
o
1
Yield (0.27 g, 66 %) as yellow crystal with m.p. 137 -138 C. H NMR (400 MHz, DMSO-d6) δ
(ppm): 0.82 (3H, d, J = 2.0 Hz, CH(CH3)), 0.84 (3H, d, J = 2.0 Hz, CH(CH3)), 1.78 – 1.90 (1H, m,
CH(CH3)2), 2.37 (3H, s, COCH3), 2.62 (3H, s, CH3), 3.86 (1H, dd, J = 10.8, 6.4 Hz, COOCH2), 3.90
(1H, dd, J = 10.8, 6.4 Hz, COOCH2), 6.00 (2H, s, OCH2O), 6.39 (1H, s, pyrimidine 4-H), 6.68 (1H,
d, J = 8.0 Hz, 6-H), 6.70 (1H, s, 4-H), 6.87 (1H, d, J = 8.0 Hz, 7-H), 11.72 (1H, s, 1-NH). 13C NMR
(100 MHz, DMSO) δ (ppm): 16.86, 19.29, 19.32, 27.58, 27.70, 52.78, 70.54, 101.69, 107.19, 107.58,
108.64, 120.35, 132.96, 145.60, 147.41, 147.92, 165.12, 173.41, 178.28. EI-MS: m/z: 390.29 [M+].
Anal. Calcd. for C19H22N2O5S: C, 58.44; H, 5.69; N, 7.18; S, 8.21. Found: C, 58.11; H, 5.65; N, 7.44;
S, 8.41.
24

5.1.5.3. (±)-2-Methoxyethyl 3-acetyl-4-(benzo[d][1,3]dioxol-5-yl)-6-methyl-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (9c)
o
1
Yield (0.30 g, 73 %) as yellow crystal with m.p. 147 -148 C. H NMR (400 MHz, DMSO-d6) δ
(ppm): 2.35 (3H, s, CH3), 2.63 (3H, s, COCH3), 3.24 (3H, s, OCH3), 3.52-3.55 (2H, m, CH2OCH3),
4.16-4.21 (1H, m, COOCH2), 4.25-4.30 (1H, m, COOCH2), 6.00 (2H, s, OCH2O), 6.33 (1H, s,
pyrimidine 4-H), 6.70 (1H, dd, J = 8.0, 1.2 Hz, 6-H), 6.72 (1H, d, J = 1.2 Hz, 4-H), 6.87 (1H, d, J =
8.0 Hz, 7-H), 11.71 (1H, s, 1-NH). 13C NMR (100 MHz, DMSO) δ (ppm): 16.92, 27.66, 53.03, 58.53,
63.76, 70.23, 101.66, 107.10, 107.59, 108.61, 120.05, 133.24, 145.61, 147.35, 147.91, 165.15,
173.57, 178.35. EI-MS: m/z: 392.28 [M+]. Anal. Calcd. for C18H20N2O6S: C, 55.09; H, 5.15; N, 7.14;
S, 8.17. Found: C, 55.19; H, 5.09; N, 7.35; S, 8.37.
5.1.5.4. (±)-Benzyl 3-acetyl-4-(benzo[d][1,3]dioxol-5-yl)-6-methyl-2-thioxo-1,2,3,4-tetrahydro-
pyrimidine-5-carboxylate (9d)
Yield (0.26 g, 59%) as yellow crystal with m.p. 169-170 oC. 1H NMR (400 MHz, DMSO-d6) δ (ppm):
2.37 (3H, s, CH3), 2.62 (3H, s, COCH3), 5.15 (1H, d, J = 12.8 Hz, COOCH2), 5.22 (1H, d, J = 12.8
Hz, COOCH2), 6.00 (2H, s, OCH2O), 6.39 (1H, s, pyrimidine 4-H), 6.67 (1H, d, J = 8.0 Hz, 6-H),
6.69 (1H, s, 4-H), 6.85 (1H, d, J = 8.0 Hz, 7-H), 7.28 – 7.35 (5H, m, Bn-H5), 11.74 (1H, s, 1-NH).
13C NMR (100 MHz, DMSO) δ (ppm): 16.97, 27.61, 52.91, 66.21, 101.70, 107.22, 107.31, 108.64,
120.32, 128.22, 128.48, 128.90, 133.03, 136.55, 145.99, 147.44, 147.94, 164.97, 173.49, 178.30. EI-
MS: m/z: 424.18 [M+]. Anal. Calcd. for C22H20N2O5S: C, 62.25; H, 4.76; N, 6.60; S, 7.56. Found: C,
62.40; H, 5.01; N, 6.79, S, 7.75.
5.1.6. General procedure for preparation of DHPM compounds (10a–d, 11a–b, and 12)
A mixture of the corresponding β-diketones or dimedone (40 mmol), compound 4 (25 mmol),
thiourea 7a / urea 7b (50 mmol), citric acid (25 mmol) and triethylorthoformate (50 mmol) was heated
under reflux for 16 h. The reaction was quenched by addition of dist. water (15 ml) and the mixture
was then cooled in an ice-bath. The precipitated solid was filtered, and recrystallized from
DMF/Acetone to give the corresponding products (10a–d, 11a–b, and 12).
5.1.6.1. (±)-5-Acetyl-4-(benzo[d][1,3]dioxol-5-yl)-6-methyl-3,4-dihydropyrimidin-2(1H)-thione
(10a)
o
o
1
Yield (0.17 g, 56%) as white powder with m.p. 250-251 C, (Lit. m.p. 238-240 C) [56]. H NMR
(400 MHz, DMSO-d6) δ (ppm): 2.14 (3H, s, CH3), 2.33 (3H, s, COCH3), 5.21 (1H, s, pyrimidine 4-
H), 6.00 (2H, s, OCH2O), 6.69 (1H, d, J = 6.4 Hz, 6-H), 6.76 (1H, s, 4-H), 6.88 (1H, d, J = 6.4 Hz,
7-H), 9.73 (1H, s, 3-NH), 10.29 (1H, s, 1-NH). 13C NMR (100 MHz, DMSO) δ (ppm): 18.69, 30.79,
53.97, 101.57, 107.45, 108.68, 110.74, 120.32, 137.32, 145.03, 147.22, 147,94, 174.31, 195.37. EI-
MS: m/z: 290.03 [M+]. Anal. Calcd. for C14H14N2O3S: C, 57.91; H, 4.87; N, 9.65; S, 11.05; Found:
C, 57.85; H, 4.66; N, 9.42; S, 11.12.
25

5.1.6.2. (±)-(4-(Benzo[d][1,3]dioxol-5-yl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)
(phenyl) methanone (10b)
Yield (0.25 g, 68 %) as yellow powder with m.p. 185-187 oC. 1H NMR (400 MHz, DMSO-d6: DMF
mixture) δ (ppm): 1.73 (3H, s, CH3), 5.21 (1H, s, pyrimidine 4-H), 6.00 (2H, s, OCH2O), 6.64 (1H,
d, J = 8.0 Hz, 6-H), 6.72 (1H, s, 4-H), 6.86 (1H, d, J = 8.0 Hz, 7-H), 7.44 – 7.56 (5H, m, C6H5CO),
13
9.65 (1H, s, 3-NH) , 10.36 (1H, s, 1-NH). C NMR (100 MHz, DMSO: DMF mixture) δ (ppm):
18.39, 55.50, 101.57, 107.17, 108.69, 110.60, 120.18, 128.41, 129.16, 132.41, 137.44, 140.54,
142.19, 147.18, 147.91, 174.46, 194.99. EI-MS: m/z (%): 352.25 [M+]. Anal. Calcd. for
C19H16N2O3S: C, 64.75; H, 4.59; N, 7.95; S, 9.10. Found: C, 64.47; H, 4.78; N, 8.21; S, 9.40.
5.1.6.3. (±)-5-Acetyl-4-(benzo[d][1,3]dioxol-5-yl)-6-methyl-3,4-dihydropyrimidin-2(1H)-one (10c)
Yield (0.14 g, 49%) as faint brown powder with m.p. 260-262 oC, (Lit. m.p. 262 oC) [56]. 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 2.09 (3H, s, CH3), 2.28 (3H, s, COCH3), 5.18 (1H, s, pyrimidine 4-
H), 5.99 (2H, s, OCH2O), 6.70 (1H, d, J = 8.0 Hz, 6-H), 6.78 (1H, s, 4-H), 6.86 (1H, d, J = 8.0 Hz,
7-H), 7.78 (1H, s, 3-NH), 9.19 (1H, s, 1-NH). 13C NMR (100 MHz, DMSO) δ (ppm): 19.34, 30.69,
54.02, 101.44, 107.39, 108.58, 109.85, 119.99, 138.70, 146.90, 147.84, 148,58, 152.50, 194.84. EI-
MS: m/z: 273.97 [M+]. Anal. Calcd. for C14H14N2O4: C, 61.31; H, 5.16; N, 10.22; Found: C, 61.13;
H, 5.31; N, 10.36.
5.1.6.4. (±)-(4-(Benzo[d][1,3]dioxol-5-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidin-5-yl)
(phenyl) methanone (10d)
Yield (0.15 g, 43 %) as yellow crystals with m.p. 214-215 oC. 1H NMR (400 MHz, DMSO-d6: DMF
mixture) δ (ppm): 1.68 (3H, s, CH3), 5.23 (1H, s, pyrimidine 4-H), 5.98 (2H, s, OCH2O), 6.66 (1H,
dd, J = 8.0, 1.4 Hz, 6-H), 6.74 (1H, d, J = 1.4 Hz, 4-H), 6.83 (1H, d, J = 8.0 Hz, 7-H), 7.42 – 7.54
13
(5H, m, C6H5CO), 7.77 (1H, s, 3-NH), 9.19 (1H, s, 1-NH). C NMR (100 MHz, DMSO: DMF
mixture) δ (ppm): 18.98, 55.41, 101.42, 107.11, 108.53, 109.84, 119.88, 128.22, 129.04, 131.92,
138.77, 141.45, 145.96, 146.83, 147.77, 152.56, 194.76. EI-MS: m/z (%): 336.24 [M+]. Anal. Calcd.
for C19H16N2O4: C, 67.85; H, 4.80; N, 8.33. Found: C, 67.56; H, 4.61; N, 8.11.
5.1.6.5. (±)-4-(Benzo[d][1,3]dioxol-5-yl)-2-thioxo-1,2,3,4-tetrahydro-5H-indeno[1,2-d]-
pyrimidin-5-one (11a)
Yield (0.19 g, 54 %) as yellow crystals with m.p. 214-215 oC (Lit. m.p. 220-222 oC) [57]. 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 6.22 (2H, s, OCH2O), 7.15 (1H, d, J = 8.0 Hz, 7-H), 7.79 (1H, s,
pyrimidine 4-H), 7.93 – 8.01 (7H, m, indene 6,7,8,9-H4 + 6-H + 1-NH + 3-NH), 8.54 (1H, d, J = 1.2
13
Hz, 4-H). C NMR (100 MHz, DMSO) δ (ppm): 102.94, 109.29, 112.32, 123.35, 123.46, 127.06,
128.03, 133.62, 136.14, 136.26, 139.75, 142.24, 146.42, 148.22, 152.78, 189.52, 190.19. EI-MS: m/z:
336.87 [M+]. Anal. Calcd. for C18H12N2O3S: C, 64.27; H, 3.60; N, 8.33; S, 9.53. Found: C, 64.35; H,
3.51; N, 8.23; S, 9.46.
26

5.1.6.6. (±)-4-(Benzo[d][1,3]dioxol-5-yl)-3,4-dihydro-1H-indeno[1,2-d]pyrimidine-2,5-dione
(11b)
Yield (0.17 g, 51 %) as yellow crystals with m.p. 210-211 oC (Lit. m.p. 217-219 oC) [57]. 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 6.22 (2H, s, OCH2O), 7.14 (1H, d, J = 8.0 Hz, 7-H), 7.78 (1H, s,
pyrimidine 4-H), 7.93 – 8.01 (7H, m, indene 6,7,8,9-H4 + 6-H + 1-NH + 3-NH), 8.53 (1H, d, J = 1.2
13
Hz, 4-H). C NMR (100 MHz, DMSO) δ (ppm): 102.94, 109.30, 112.32, 123.36, 123.47, 127.06,
128.03, 133.63, 136.15, 136.27, 139.75, 142.24, 146.42, 148.23, 152.79, 189.53, 190.20. EI-MS: m/z:
319.98 [M+]. Anal. Calcd. for C18H12N2O4: C, 67.50; H, 3.78; N, 8.75. Found: C, 67.75; H, 3.88; N,
8.94.
5.1.6.7. (±)-4-(Benzo[d][1,3]dioxol-5-yl)-7,7-dimethyl-4,6,7,8-tetrahydroquinazoline-2,5(1H,3H)-
dione (12)
Yield (0.19 g, 58 %) as faint yellow powder with m.p. 178 -179 oC. 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 0.92 (3H, s, CH3), 1.02 (3H, s, CH3), 2.05 (1H, d, J = 16.0 Hz, quinazoline 6-H), 2.19 (1H,
d, J = 16.0 Hz, quinazoline 6-H), 2.29 (1H, d, J = 17.2 Hz, quinazoline 8-H), 2.40 (1H, d, J = 17.2
Hz, quinazoline 8-H), 5.08 (1H, s, quinazoline 4-H), 5.98 (2H, s, OCH2O), 6.70 (1H, dd, J = 8.0, 1.3
Hz, 6-H), 6.73 (1H, d, J = 1.3 Hz, 4-H), 6.85 (1H, d, J = 8.0 Hz, 7-H), 7.74 (1H, s, 3-NH) , 9.48 (1H,
s, 1-NH). 13C NMR (100 MHz, DMSO) δ (ppm): 27.42, 29.14, 32.77, 39.49, 50.27, 52.11, 101.37,
107.11, 107.85, 108.47, 119.79, 139.17, 146.68, 147.61, 152.32, 152.84, 193.44. EI-MS: m/z: 314.03
[M+]. Anal. Calcd. for C17H18N2O4: C, 64.95; H, 5.78; N, 8.91. Found: C, 64.83; H, 5.52; N, 8.78.
5.1.7. General procedure for preparation of compounds (15 and 16)
A mixture of compound 4 (10 mmol), 2- aminobenzimidazole 14 (10 mmol) and acetyl acetone /
benzyl acetoacetate (10 mmol) in DMF (30 ml) was heated under reflux for 10 hours. After cooling
to room temperature, acetone (15 ml) was added. The reaction mixture was permitted to stand
overnight and then filtered to yield precipitated solid of product, which was recrystallized from
DMF/acetone.
5.1.7.1. (±)-Benzyl4-(benzo[d][1,3]dioxol-5-yl)-2-methyl-1,4-dihydrobenzo[4,5]imidazo[1,2-a]
pyrimidine -3-carboxylate (15)
Yield (0.38 g, 83%) as white powder with m.p. 258-260 oC. 1H NMR (400 MHz, DMSO-d6) δ (ppm):
2.48 (3H, s, CH3), 5.05 (1H, d, J = 12.8 Hz, COOCH2), 5.16 (1H, d, J = 12.8 Hz, COOCH2), 5.94
(2H, s, OCH2O), 6.41 (1H, s, pyrimidine 4-H), 6.75 (1H, d, J = 8.0 Hz, 7-H), 6.84 (1H, d, J = 8.0 Hz,
6-H), 6.88 (1H, s, 4-H), 6.98 (1H, t, J = 7.2 Hz, benzimidazole 5-H), 7.05 (1H, t, J = 7.2 Hz,
benzimidazole 6-H), 7.21 (2H, d, J = 7.2 Hz, benzimidazole 4,7-H2), 7.31 – 7.36 (5H, m, Bn-H5),
10.90 (1H, br s, 1-NH). 13C NMR (100 MHz, DMSO) δ (ppm): 19.30, 56.03, 65.41, 98.00, 101.55,
108.08, 108.44, 110.65, 117.25, 120.67, 121.15, 122.24, 128.12, 128.19, 128.72, 131.94, 136.29,
136.96, 142.72, 145.85, 147.20, 147.68, 165.48. EI-MS: m/z: 439.20 [M+]. Anal. Calcd. for
27

C26H21N3O4: C, 71.06; H, 4.83; N, 9.56; Found: C, 71.29; H, 4.67; N, 9.72.
5.1.7.2. (±)-1-(4-(Benzo[d][1,3]dioxol-5-yl)-2-methyl-1,4-dihydrobenzo[4,5]imidazo[1,2-a]
pyrimidin-3-yl)ethan-1-one (16)
Yield (0.20 g, 55%) as white powder with m.p. 250-252 oC. 1H NMR (400 MHz, DMSO-d6) δ (ppm):
2.24 (3H, s, CH3), 2.48 (3H, s, COCH3), 5.93 (2H, s, OCH2O), 6.54 (1H, s, pyrimidine 4-H), 6.80
(1H, d, J = 8.0 Hz, 6-H), 6.94 (1H, d, J = 8.0 Hz, 7-H), 6.95 (1H, s, 4-H), 7.02 (1H, t, J = 7.6 Hz,
benzimidazole 5-H), 7.06 (1H, t, J = 7.6 Hz, benzimidazole 6-H), 7.35 (1H, d, J = 7.6 Hz,
13
benzimidazole 4-H), 7.47 (1H, d, J = 7.6 Hz, benzimidazole 7-H), 10.80 (1H, s, 1-NH). C NMR
(100 MHz, DMSO) δ (ppm): 20.19, 31.11, 55.83, 101.58, 107.98, 108.53, 109.09, 110.67, 117.31,
120.65, 121.24, 122.27, 132.02, 136.02, 142.75, 145.89, 146,57, 147.26, 147.84, 194.78. EI-MS: m/z:
347.59 [M+]. Anal. Calcd. for C20H17N3O3: C, 69.15; H, 4.94; N, 12.09; Found: C, 69.42; H, 5.21; N,
11.93.
5.2. Biology
5.2.1. Preliminary in vitro anticancer screening [58]
The human tumor cell lines of the cancer screening panel are grown in RPMI 1640 medium
containing 5% fetal bovine serum and 2 mM L-glutamine. For a screening experiment, cells are
injected into 96 well micro titer plates in 100 μl at plating densities ranging from 5,000 to 40,000
cells/well depending on the doubling time of separate cell lines. After cell inoculation, incubation of
the micro titer plates was done at 37° C, 5 % CO2, 95 % air and 100 % relative humidity overnight
prior to adding of experimental drugs. After 24 h, each cell line of two plates are fixed in situ with
TCA, to represent a calculation of the cell population for each cell line at the time of drug addition.
Experimental drugs are solubilized in DMSO at 400-fold the final maximum test concentration and
kept frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate is thawed and
diluted to twice the wanted final maximum test concentration with complete medium containing 50
μg/ml gentamicin. Aliquots of 100 μl of these different dilutions of drug are added to the suitable
microtiter wells previously containing 100 μl of medium, leading to the required final drug
concentrations 10 μM. Controls, containing only DMSO and phosphate buffer saline at identical
dilutions, were done in the same method. Following drug addition, the plates are incubated for an
extra 48 h at 37°C, 5 % CO2, 95 % air, and 100 % relative humidity. For adherent cells, the assay is
finished by the addition of cold TCA. Cells are fixed in situ by the mild addition of 50 μl of cold 50%
(w/v) TCA (final concentration, 10 % TCA) and incubated for 60 minutes at 4°C.
The supernatant is removed, and the plates are washed several times with dist. water and air dried.
Sulforhodamine B (SRB) solution (100 μl) at 0.4 % (w/v) in 1 % acetic acid is added to each well,
and plates are incubated for ten minutes at 25oC.
28

After staining, unbound dye is discarded by washing several times with 1 % acetic acid and the plates
are air dried. Bound stain is then solubilized with 10 mM trizma base, and the absorbance is recorded
on an automated reader at a wavelength of 515 nm. For suspension cells, the methodology is the
similar except that the assay is finished by fixing settled cells at the bottom of the wells by gradually
adding 50 μl of 80 % TCA (final conc., 16 % TCA). Using the seven absorbance measurements, the
percentage growth of the treated cells is calculated compared to the untreated control cells.
5.2.2. Kinesin inhibition assay
ATPase assays were performed as previously described [23, 59] by measuring the inhibition of basal
ATPase activity of human Eg51–368 at an estimated protein concentration between 200 and 400 nm.
The Eg5 concentration was estimated by the Bradford reagent. All data were measured in triplicate,
and IC50 values are given as mean values ±SD. Detailed procedure for kinesin inhibition assay is
available in supplementary information (SI).
5.2.3. HEK-293 cell growth inhibition assay
Detailed procedure for HEK 293 cell growth inhibition assay with representative IC50 curves were
discussed in SI as previously described [60]. All data were measured in triplicate, and IC50 values are
given as mean values ±SD.
5.2.4. Cell cycle analysis and induction of apoptosis
5.2.4.1. Flow cytometric analysis of cell-cycle distribution
Cell cycle analysis was carried out through flow cytometry [61]. SNB-75 cells were treated with the
test compound, seeded and incubated for 24 hours at 37 °C, 5% CO2. Cells were washed twice with
phosphate buffer saline. Then, collection and centrifugation of cell pellets took place, followed by
fixing with 70% (v/v) ethanol in ice for 15 minutes. Pellets were collected again, where they were
incubated with propidium iodide (PI) staining solution that contains 50 µg/mL PI, 0.1 mg/mL RNAse
A and 0.05% Triton X-100. After incubation for one hour at room temperature, it was analyzed by
flow cytometry on FC500 cytometer (Beckman Coulter) and cell cycle distributions were calculated.
5.2.4.2. Analysis of cellular apoptosis
AnnexinV-fluorescein isothiocyanate (Annexin V-FITC) is a protein that possesses high affinity to
phosphatidyl serine PS, which can be detected by staining with Annexin V- (FITC) and counter
staining with propidium iodide (PI). Apoptosis was initially induced by incubation of cells in a
density of 1.2-1.8 X 104 cells/well in complete growing medium (100 µ L) with addition of the tested
compound (100 µ L) in 96-microwell plates for 24 h. Then, 1-5 X 105 cells were collected by
centrifugation and resuspended in 500 µ L of binding buffer. Annexin V-FITC (5 µ L) along with PI
(5 µ L) were added and incubated in the dark for 5 minutes at room temperature. Analysis of Annexin-
V-FITC binding was performed using FACS caliber flow cytometer (BD Biosciences, San Jose. CA).
29