Design, synthesis and antimicrobial activities of thiouracil derivatives containing triazolo-thiadiazole as SecA inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.12.053

A series of novel thiouracil derivatives containing a triazolo-thiadiazole moiety (7a-7l) have been synthesized by structural modifications on a lead SecA inhibitor, 2. All the compounds have been evaluated for their antibacterial activities against Bacillus amyloliquefaciens, Staphylococcus aureus, and Bacillus subtilis. Compounds 7d and 7g were also tested for their inhibitory activities against SecA ATPase due to their promising antimicrobial activities. The inhibitory activity of compound 7d was found to be higher than that of 2. Molecular docking work suggests that compound 7d might bind at a pocket close to the ATPase ATP-binding domain.

Full text of DOI:10.1016/j.ejmech.2016.12.053

European Journal of Medicinal Chemistry 127 (2017) 159e165
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and antimicrobial activities of thiouracil derivatives
containing triazolo-thiadiazole as SecA inhibitors
,
,
, **
Penglei Cui a b, Xiaoliu Li a *, Mengyuan Zhu c, Binghe Wang c, Jing Liu d, Hua Chen a
a Key Laboratory of Chemical Biology of Hebei Province, College of Chemistry and Environmental Science, Hebei University, Baoding 071002, China
b College of Science, Agricultural University of Hebei, Baoding 071001, China
c Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30302-4098, USA
d College of Veterinary Medicine, Agricultural University of Hebei, Baoding 071001, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel thiouracil derivatives containing a triazolo-thiadiazole moiety (7a-7l) have been syn-
thesized by structural modifications on a lead SecA inhibitor, 2. All the compounds have been evaluated
for their antibacterial activities against Bacillus amyloliquefaciens, Staphylococcus aureus, and Bacillus
subtilis. Compounds 7d and 7g were also tested for their inhibitory activities against SecA ATPase due to
their promising antimicrobial activities. The inhibitory activity of compound 7d was found to be higher
than that of 2. Molecular docking work suggests that compound 7d might bind at a pocket close to the
ATPase ATP-binding domain.
Received 13 August 2016
Received in revised form
23 December 2016
Accepted 24 December 2016
Available online 27 December 2016
Keywords:
© 2016 Elsevier Masson SAS. All rights reserved.
Antibacterial activity
SecA inhibitors
Thiouracil
Triazolo-thiadiazole
Molecular simulation
1. Introduction
translocation from the cytosol across or into the cytoplasmic
membrane [17e20]. Because SecA is a conserved and essential
Since the 1980s, bacterial drug resistance has become a major
problem because of the widespread use, even abuse, of antibiotics.
With Gram-positive bacteria, many drug resistant strains including
methicillin-resistant Staphylococcous aureurs (MRSA), methicillin-
resistant Staphylococcus epidermidis (MRSE), penicillin-resistant
Streptococcus pneumonia (PRSP) and vancomycin-resistant Entero-
cocci (VRE) are posing serious public health problems [1e4]. As a
result, there is an urgent need for new types of antibacterial drugs,
especially important is the search for new targets or mechanisms to
achieve antimicrobial effects [5e12]. Along this line [13e16], we are
interested in targeting SecA, which is a critical protein secretion
machinery essential for bacterial survival.
In bacterial cells, over 30% proteins become functional after they
are transported outside of the cytoplasm. Trans-membrane move-
ment of most of these proteins is via the Sec pathway (i.e. secretion
pathway). SecA ATPase is one of the essential components in the
Sec machinery, which provides a major pathway to help protein
protein in nearly all bacteria, it is considered as a promising anti-
bacterial drug target [21e25]. In recent years, studies have
confirmed that inhibiting SecA can lead to bacteriostatic and
bactericidal effects [26,27]. Therefore, developing innovative anti-
bacterial drugs by targeting SecA has drawn much attention. It is
expected that such inhibitors may have the intrinsic ability to
minimize side effects because there is no counterpart of SecA in
human. At present, small organic molecules that can inhibit SecA
mainly include Rose Bengal 1, thiouracil 2, and triazole 3 derivatives
(Fig. 1) [28e31]. In order to find new SecA inhibitors to better un-
derstand the SAR and the biological mechanism of SecA, it is
worthwhile to explore the chemistry space of known inhibitor to
increase structural diversity.
The compounds containing triazole or thiadiazole have a wide
range of biological activity. Such two pharmacophore were fused to
a triazolo-thiadiazole moiety, which also has a variety of biological
and pharmacological activity [32e34], such as antibacterial and
anti-tumor. Due to their strong ability to form hydrogen bond with
macro biomoleculars [35], we would like to report the design and
synthesis of compound 7 containing both thiouracil and triazolo-
thiadiazole motifs (Scheme 1). Such compounds are based on the
* Corresponding author.
** Corresponding author.
E-mail addresses: lixl@hbu.cn (X. Li), hua-todd@163.com (H. Chen).
http://dx.doi.org/10.1016/j.ejmech.2016.12.053
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

160
P. Cui et al. / European Journal of Medicinal Chemistry 127 (2017) 159e165
Fig. 1. The structures of SecA inhibitors.
Scheme 1. Synthesis of compounds 7a-7l. Conditions and reagents: (a) POCl3, reflux; (b) K2CO3, acetonitrile, reflux.
parent compound 2 (Fig. 1). We hypothesize that the triazolo-
hydrogen-bond interactions with the target protein SecA, and
thiadiazole scaffold would have the capability to form more
would be beneficial to improving the antibacterial activity.

P. Cui et al. / European Journal of Medicinal Chemistry 127 (2017) 159e165
161
2. Results and discussions
for their antibacterial activities. The activities of compounds 7j-l
improved significantly when R1 is 2,6-diCl on the phenyl near
triazolo-thiadiazole group and R2 is fixed as 2,4-dichloro, 2,6-
dichloro and 4-phenyl, respectively. The above structure activity
relationships (SAR) analysis suggested that the introduction of
additional Cl atoms on the marginal phenyls would be beneficial for
antibacterial activities. Such insights should help to guide further
design and synthesis of the more potent antibacterial agents
derived from compound 2.
2.1. Chemistry
In this study, a series of novel thiouracil derivatives (7a-l) con-
taining a triazolo-thiadiazole moiety were synthesized. The syn-
thetic route for 7 is shown in Scheme 1. The reaction of aromatic
aldehydes, cyano ethyl acetate and thiourea with pyridine as
catalyst in absolute ethanol at reflux temperature for 8 h afforded
thiouracil 6 [28]. Intermediate 4, prepared by using aromatic acids
as starting materials through multi-step reactions [30], was reacted
with 4-(chloromethyl)benzoyl chloride in POCl3 to give compound
5. The target compounds 7 were obtained by reacting 5 and thio-
uracil 6 in acetonitrile at reflux temperature.
2.3. SecA inhibitory activity
Two potent compounds 7d and 7g with relatively high anti-
bacterial activities were chosen for evaluation SecA inhibitory ac-
tivities [28e31]. The results indicated that compound 7d showed
significant inhibitory activity as shown in Fig. 2. For example, the
2.2. Antibacterial activity
inhibitory activity of 7d was 51% at 10
The IC50 (50% inhibitory concentrations) value of compound 7d was
calculated as 9.7 g/mL, which is lower than that of compound 2
(20.8 g/mL). Unfortunately, the IC50 value of compound 7g was not
mg/mL and 72% at 20 mg/mL.
All the newly synthesized compounds were evaluated for their
antibacterial activities against three strains of Gram-positive or-
ganisms including Bacillus amyloliquefaciens, Staphylococcus aureus,
and Bacillus subtilis [36,37]. Norfloxacin and the parent compound 2
(Fig. 1) were used as the positive controls. The tests were carried
out in accordance with the plate colony counting protocol. The
m
m
accurately measured due to its very poor solubility under assay
conditions.
2.4. Computational modeling
results are shown in Table 1 (the concentration is 25 mg/mL). The
results show that compounds 7 (a-l) have good to potent inhibitory
activity against the three tested bacterial strains. Among them,
compounds 7d and 7g have the strongest antibacterial activities
against B. subtilis and their inhibitory rates were above 90%, higher
than that of approved drug norfloxacin. Their inhibitory rates
exceeded 85% against S. aureus, similar with that of norfloxacin.
Although their anti-B. amyloliquefaciens activities were less than
that of norfloxacin, their inhibitory rates were also above 80%,
which suggested that both compounds had potent antibacterial
activities. Compounds 7i-7l also had broad antibacterial activities
against the tested three organisms with above 80% inhibition
except 7j against B. amyloliquefaciens, similar with that of nor-
floxacin against B subtilis. The anti B. subtilis activities of com-
pounds 7d, 7g and 7i-l were superior to that of compound 2, a
known SecA inhibitor as antibacterial agent. Additionally, the ac-
tivities of 7i and 7k against B. amyloliquefaciens, 7d against
S. aureus, and 7h against B. subtilis were slightly higher than that of
2. Such results indicate that the introduction of the triazolo-
thiadiazole pharmacophore to 2 could increase and broaden its
antibacterial activity. When R1 is H or 2-Cl, compound 7d and 7g
(R2 ¼ 2,4-dichloro) have relatively higher antibacterial activities
than those of the tested compounds, such results indicate that two
Cl atoms on the phenyl ring near thiouracil seemed to be favorable
In order to achieve some initial insights into the binding in-
teractions, the newly synthesized compound 7d was docked into
the SecA crystal structure. Two possible binding pockets were
selected: the ATPase ATP-binding site and the pocket between IRA2
and NBD domain [38]. The second pocket is close to the ATP binding
site as we proposed before (the closest distance of ATP and docked
Fig. 2. SecA inhibitory activities of 7d and 2.
Table 1
Antibacterial activities of 7a-7l (Inhibitory rate/% at 25 mg/mL).
Compounds
R1
R2
Bacillus amyloliquefaciens
Staphylococcus aureus
Bacillus subtilis
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
H
H
H
H
H
H
2-Cl
2-Cl
H
2-Cl
4-Cl
57%
66%
62%
82%
60%
58%
80%
78%
81%
78%
86%
82%
85%
89%
44%
46%
49%
88%
56%
68%
85%
56%
83%
81%
82%
84%
87%
86%
48%
55%
51%
90%
49%
57%
92%
63%
88%
86%
84%
86%
62%
85%
2,4-dichloro
2,6-dichloro
4-Ph
2,4-dichloro
2,6-dichloro
4-Ph
2,4-dichloro
2,6-dichloro
4-Ph
2-Cl
2,6-dichloro
2,6-dichloro
2,6-dichloro
2
Norfloxacin

162
P. Cui et al. / European Journal of Medicinal Chemistry 127 (2017) 159e165
molecule is 9.3 Å) [31]. 20 outputs were generated and the docking
scores 3.56e5.12 at the ATP-binding site and 4.62e5.97 at pocket
that was identified for the lead structures [31]. Thus it is reasonable
to suggest that the new inhibitor binds at a similar position as the
lead structure. The interactions are shown in Fig. 3: molecule 7d
generated hydrogen-bond interactions with ARG566, GLN570,
ARG642 and two water molecules. It also forms cation-pi in-
teractions with ARG642 and hydrophobic interaction with VAL131
and ILE216.
4.2. General procedure for the synthesis of compounds 7(a-l)
To a solution of aromatic acid (10 mmol) in absolute ethanol
(50 mL) was added SOCl2 (15 mmol); then the mixture was heated
at 60 ꢁC for 3 h. Upon completion, the reaction mixture was cooled
to ambient temperature and the solvent was removed in vacuo. The
residue was dissolved in ethyl acetate and washed with saturated
aqueous sodium bicarbonate (10 mL) and water (10 mL ꢂ 2), then
the organic layer was dried with sodium sulfate for 2 h. After
filtration, the solvent was removed in vacuo to give the aromatic
ester. The crude ester (5 mmol) and 30 mL 80% hydrazine hydrate
were added into a 100 mL flask; then the mixture were refluxed for
about 5 h. After completion of the reaction as monitored by TLC, the
mixture was cooled to room temperature, and precipitate was
filtered and dried to give the aryl hydrazide product. To a solution of
KOH (6 mmol) in absolute ethanol (40 mL) was added the aryl
hydrazide (3 mmol). Then CS2 (10 mL) was added into the mixture
dropwise, leading to light yellow precipitates, which were filtered
and dried to give the intermediate. A solution of this intermediate
(2 mmol) in hydrazine hydrate (20 mL) were heated under reflux
for about 4 h. Upon completion, the mixture was poured into cold
water (150 mL). Then the aqueous solution was acidified to pH ~5
by slow addition of 1 M HCl. This caused the formation of pre-
cipitates, which were filtered using vacuum filtration to give
compound 4.
3. Conclusions
A series of novel thiouracil derivatives containing a triazolo-
thiadiazole moiety 7 (a-l) were designed, synthesized, and evalu-
ated for their antibacterial activities. The results showed that most
of the compounds have strong antibacterial activity. Additionally,
compound 7d expressed better inhibitory activity against SecA
ATPase than that of the known inhibitor 2, suggesting a beneficial
role for the newly introduced triazole-thiadiazole moiety. Molec-
ular simulations suggest that compound 7 might bind to a pocket
close to the ATPase ATP-binding domain.
A solution of compound 4 (1 mmol) and 4-(chloromethyl)
benzoyl chloride (1.2 mmol) in POCl3 (30 mL) were heated under
reflux for about 5 h. Upon completion, the solvent was removed in
vacuo and then 30 mL water was added into the flask. The solution
was neutralized by slow addition of 1 M HCl, leading to formation of
precipitates, which were filtered using vacuum filtration to give
compound 5.
To a solution of aromatic aldehyde (10 mmol), ethyl cyanoace-
tate (1.0 mL, 10 mmol), and thiourea (0.76 g, 10 mmol) in absolute
ethanol (50 mL) was added piperidine (2.0 mL, 20 mmol); the
mixture was heated under reflux overnight and then cooled to
room temperature. The precipitate was dissolved in 0.5 M NaOH
(20 mL) and washed with ethyl acetate (10 mL ꢂ 3). The aqueous
solution was then acidified to pH 2 by slow addition of 1 M HCl.
Then the product precipitated (crystallized) out to give compound
6.
4. Experimental section
4.1. General
Column chromatography was carried out on flash silica gel
(300e800 mesh). TLC analysis was conducted on silica gel plates
(Silica G UV254). Melting points were measured in an open capil-
lary on a SGW X-4 melting point apparatus and are uncorrected.
Element analysis was performed using a Heraeus (CHNO, rapid)
elemental analyzer. IR spectra were determined on a WQF-510 as
KBr tablets for solid samples and were expressed in cmꢀ1 scale.
NMR spectra were recorded at 600 MHz for 1H on a Bruker in-
strument. Chemical shifts (
d values) and coupling constants (J
values) are given in ppm and hertz, respectively, using TMS (1H
NMR) solvents as internal standards. ESI-MS spectra were deter-
mined using an Agilent G6300 ion Trap mass spectrometer, and
signals were recorded in m/z. Absorption spectra were recorded on
a FLUOstar Omega MicroplateReader.
A solution of compound 5 (1.1 mmol), compound 6 (1 mmol)
and K2CO3 (3 mmol) in acetonitrile (30 mL) were heated under
Fig. 3. The proposed docking conformation of 7d within the pocket between IRA2 and NBD domain. The crystal structure is shown in cyan ribbon and sticks; 7d is shown in green
sticks; and water is shown in red spheres. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

P. Cui et al. / European Journal of Medicinal Chemistry 127 (2017) 159e165
163
reflux. After the completion of the reaction as indicated by TLC, the
2.50; N, 16.57.
solvent was evaporated under vacuum to obtain crude 7, which was
washed with 0.2 mol/L NaOH and dried in vacuo. Subsequent pu-
rification using column chromatography (ethyl acetate/
methanol ¼ 15:1) gave the target products 7a-l.
4.2.6. 4-Biphenyl-4-yl-6-oxo-2-[4-(3-phenyl- [1,2,4]triazolo [3,4-b]
[1,3,4]thiadiazol-6-yl)-benzylsulfanyl]-1,6-dihydro-pyrimidine -5-
carbonitrile (7f)
Light yellow solid, Yield 59%; mp 232e235 ꢁC; IR (KBr, , cmꢀ1):
n
4.2.1. 6-Oxo-4-phenyl-2-[4-(3-phenyl- [1,2,4]triazolo [3,4-b] [1,3,4]
thiadiazol-6-yl)-benzylsulfanyl]-1,6-dihydropyrimidine -5-
carbonitrile (7a)
3360 (NeH), 2360 (eCN) and 1654 (C]O); 1H NMR (600 MHz,
DMSO-d6)
d
(ppm): 8.33 (d, J ¼ 7.2 Hz, 2H, ArH), 8.02 (d, J ¼ 8.4 Hz,
2H, ArH), 7.89 (d, J ¼ 8.4 Hz, 2H, ArH), 7.78 (d, J ¼ 7.8 Hz, 2H, ArH),
7.74 (d, J ¼ 7.8 Hz, 2H, ArH), 7.71 (d, J ¼ 8.4 Hz, 2H, ArH), 7.64 (t,
J ¼ 7.2 Hz, 2H, ArH), 7.58 (t, J ¼ 7.8 Hz, 1H, ArH), 7.49 (t, J ¼ 7.2 Hz,
2H, ArH), 7.40 (t, J ¼ 7.8 Hz,1H, ArH), 4.42 (s, 2H, CH2); MS (ESI) m/z:
595.1 ([M - H]þ). Anal. Calcd for C33H21N7OS2: C, 66.54; H, 3.55; N,
16.46. Found: C, 66.46; H, 3.50; N, 16.38.
Light yellow solid, Yield 55%; mp 225e228 ꢁC; IR (KBr, , cmꢀ1):
n
3390 (NeH), 2364 (eCN) and 1652 (C]O); 1H NMR (600 MHz,
DMSO-d6)
d
(ppm): 8.33 (d, J ¼ 7.2 Hz, 2H, ArH), 7.99 (d, J ¼ 7.8 Hz,
2H, ArH), 7.75 (t, J ¼ 3.6 Hz, 2H, ArH), 7.69 (d, J ¼ 8.4 Hz, 2H, ArH),
7.63 (t, J ¼ 7.8 Hz, 2H, ArH), 7.57 (t, J ¼ 7.8 Hz, 1H, ArH), 7.47 (t,
J ¼ 3.6 Hz, 3H, ArH), 4.40 (s, 2H, CH2); MS (ESI) m/z: 518.2 ([M - H]þ).
Anal. Calcd. for C27H17N7OS2: C, 62.41; H, 3.30; N, 18.87. Found: C,
62.34; H, 3.25; N, 18.81.
4.2.7. 2-{4-[3-(2-Chloro-phenyl)- [1,2,4]triazolo [3,4-b] [1,3,4]
thiadiazol-6-yl]-benzylsulfanyl}-4-(2,4-dichloro-phenyl)-6-oxo-1,6-
dihydro-pyrimidine-5-carbonitrile (7g)
4.2.2. 4-(2-Chloro-phenyl)-6-oxo-2-[4-(3-phenyl- [1,2,4] triazolo
[3,4-b] [1,3,4]thiadiazol-6-yl)-benzylsulfanyl]-1,6-dihydro-
pyrimidine-5-carbonitrile (7b)
Light yellow solid, Yield 65%; mp 201e204 ꢁC; IR (KBr, , cmꢀ1):
n
3360 (NeH), 2360 (eCN) and 1640 (C]O); 1H NMR (600 MHz,
DMSO-d6)
d
(ppm): 7.98 (d, J ¼ 7.2 Hz, 2H, ArH), 7.90 (d, J ¼ 7.8 Hz,
Light yellow solid, Yield 65%; mp 204e206 ꢁC; IR (KBr,
n
, cmꢀ1):
2H, ArH), 7.75 (t, J ¼ 3.6 Hz, 2H, ArH), 7.69 (d, J ¼ 8.4 Hz, 1H, ArH),
7.63 (t, J ¼ 7.8 Hz, 2H, ArH), 7.57 (d, J ¼ 7.8 Hz, 1H, ArH), 7.47 (t,
J ¼ 3.6 Hz,1H, ArH), 4.40 (s, 2H, CH2); 13C NMR (150 MHz, DMSO-d6)
3380 (NeH), 2362 (eCN) and 1652 (C]O); 1H NMR (600 MHz,
CD3OD)
d
(ppm): 8.08 (d, J ¼ 7.2 Hz, 2H, ArH), 7.99 (d, J ¼ 7.8 Hz, 2H,
ArH), 7.75 (t, J ¼ 3.6 Hz, 2H, ArH), 7.69 (d, J ¼ 8.4 Hz, 2H, ArH), 7.63
(d, J ¼ 7.8 Hz, 2H, ArH), 7.57 (t, J ¼ 7.2 Hz, 1H, ArH), 7.47 (t, J ¼ 3.6 Hz,
2H, ArH), 4.40 (s, 2H, CH2); MS (ESI) m/z: 551.3 ([M - 3H]þ). Anal.
Calcd for C27H16ClN7OS2: C, 58.53; H, 2.91; N,17.70. Found: C, 58.60;
H, 2.85; N, 17.61.
d (ppm): 177.6, 159.3, 137.3, 132.1, 131.9, 131.7, 130.0, 129.2, 128.9,
117.1, 115.7, 113.9, 52.5; MS (ESI) m/z: 622.0 ([M - H]þ). Anal. Calcd.
for C27H14Cl3N7OS2: C, 52.06; H, 2.27; N, 15.74. Found: C, 52.14; H,
2.20; N, 15.61.
4.2.8. 2-{4-[3-(2-Chloro-phenyl)- [1,2,4]triazolo [3,4-b] [1,3,4]
thiadiazol-6-yl]-benzylsulfanyl}-4-(2,6-dichloro-phenyl)-6-oxo-1,6-
dihydro-pyrimidine-5-carbonitrile (7h)
4.2.3. 4-(4-Chloro-phenyl)-6-oxo-2-[4-(3-phenyl- [1,2,4] triazolo
[3,4-b] [1,3,4]thiadiazol-6-yl)-benzylsulfanyl]-1,6-dihydro-
pyrimidine-5-carbonitrile (7c)
Light yellow solid, Yield 65%, mp 224e227 ꢁC; IR (KBr, , cmꢀ1):
n
Light yellow solid, Yield 65%; mp 220e223 ꢁC; IR (KBr,
n
, cmꢀ1):
3370 (NeH), 2360 (eCN) and 1664 (C]O); 1H NMR (600 MHz,
DMSO-d6)
3372 (NeH), 2360 (eCN) and 1662 (C]O); 1H NMR (600 MHz,
CD3OD)
d
(ppm): 7.99 (d, J ¼ 7.8 Hz, 2H, ArH), 7.88 (d, J ¼ 7.8 Hz,
d
(ppm): 8.04 (d, J ¼ 7.2 Hz, 2H, ArH), 7.97 (d, J ¼ 7.8 Hz, 2H,
2H, ArH), 7.74 (t, J ¼ 3.6 Hz, 2H, ArH), 7.66 (d, J ¼ 7.8 Hz, 1H, ArH),
7.60 (t, J ¼ 7.2 Hz, 2H, ArH), 7.54 (d, J ¼ 7.8 Hz, 1H, ArH), 7.43 (t,
J ¼ 4.8 Hz,1H, ArH), 4.41 (s, 2H, CH2); MS (ESI) m/z: 622.1 ([M - H]þ).
Anal. Calcd. for C27H14Cl3N7OS2: C, 52.06; H, 2.27; N, 15.74. Found:
C, 52.00; H, 2.32; N, 15.67.
ArH), 7.78 (t, J ¼ 3.6 Hz, 2H, ArH), 7.71 (d, J ¼ 8.4 Hz, 2H, ArH), 7.64
(d, J ¼ 7.2 Hz, 2H, ArH), 7.55 (t, J ¼ 7.8 Hz,1H, ArH), 7.47 (t, J ¼ 3.6 Hz,
2H, ArH), 4.40 (s, 2H, CH2); MS (ESI) m/z: 553.4 ([M - H]þ). Anal.
Calcd for C27H16ClN7OS2: C, 58.53; H, 2.91; N,17.70. Found: C, 58.59;
H, 2.82; N, 17.61.
4.2.9. 4-Biphenyl-4-yl-2-{4-[3-(2-chloro-phenyl)- [1,2,4] triazolo
[3,4-b] [1,3,4]thiadiazol-6-yl]-benzylsulfanyl}-6-oxo-1,6-dihydro-
pyrimidine-5-carbonitrile (7i)
4.2.4. 4-(2,4-Dichloro-phenyl)-6-oxo-2-[4-(3-phenyl- [1,2,4]
triazolo [3,4-b] [1,3,4]thiadiazol-6-yl)-benzylsulfanyl]-1,6-dihydro-
pyrimidine-5-carbonitrile (7d)
Light yellow solid, Yield 65%, mp 231e234 ꢁC; IR (KBr, , cmꢀ1):
n
Light yellow solid, Yield 54%; mp 214e217 ꢁC; IR (KBr,
n
, cmꢀ1):
3360 (NeH), 2364 (eCN) and 1662 (C]O); 1H NMR (600 MHz,
DMSO-d6)
3370 (NeH), 2360 (eCN) and 1655 (C]O); 1H NMR (600 MHz,
CD3OD)
d
(ppm): 8.01 (s, 2H, ArH), 7.92 (d, J ¼ 7.2 Hz, 4H, ArH),
d
(ppm): 7.94 (d, J ¼ 7.8 Hz, 2H, ArH), 7.79 (d, J ¼ 8.4 Hz, 2H,
7.77 (d, J ¼ 8.4 Hz, 2H, ArH), 7.69 (m, 4H, ArH), 7.48 (t, J ¼ 7.8 Hz, 2H,
ArH), 7.40 (t, J ¼ 7.2 Hz, 1H, ArH), 7.25 (m, 2H, ArH), 4.52 (s, 2H,
ArH), 7.62 (d, J ¼ 7.2 Hz, 1H, ArH), 7.55 (d, J ¼ 8.4 Hz, 2H, ArH), 7.53
(d, J ¼ 8.4 Hz, 2H, ArH), 7.30 (d, J ¼ 6.6 Hz, 1H, ArH), 7.23 (m, 2H,
CH2); MS (ESI) m/z: 629.7 ([M
-
H]þ). Anal. Calcd. for
ArH), 4.37 (s, 2H, CH2); 13C NMR (150 MHz, DMSO-d6)
d
(ppm):
C33H20ClN7OS2: C, 62.90; H, 3.20; N, 15.56. Found: C, 62.80; H, 3.16;
177.6, 159.3, 131.7, 130.1, 129.3, 128.9, 126.9, 115.5, 113.9, 52.2; MS
(ESI) m/z: 587.4 ([M - H]þ). Anal. Calcd. for C27H15Cl2N7OS2: C, 55.11;
H, 2.57; N, 16.66. Found: C, 55.19; H, 2.50; N, 16.57.
N, 15.50.
4.2.10. 4-(2,4-Dichloro-phenyl)-2-{4-[3-(2,6-dichloro-phenyl) -
[1,2,4]triazolo [3,4-b] [1,3,4]thiadiazol-6-yl]-benzylsulfanyl} -6-oxo-
1,6-dihydro-pyrimidine-5-carbonitrile (7j)
4.2.5. 4-(2,6-Dichloro-phenyl)-6-oxo-2-[4-(3-phenyl- [1,2,4]
triazolo [3,4-b] [1,3,4]thiadiazol-6-yl)-benzylsulfanyl]-1,6-dihydro-
pyrimidine-5-carbonitrile (7e)
Light yellow solid, Yield 65%; mp 217e219 ꢁC; IR (KBr, , cmꢀ1):
n
3370 (NeH), 2360 (eCN) and 1650 (C]O); 1H NMR (600 MHz,
CD3OD)
Light yellow solid, Yield 50%; mp 229e231 ꢁC; IR (KBr,
n
, cmꢀ1):
d
(ppm): 7.99 (s, 1H, ArH), 7.93 (d, J ¼ 7.8 Hz, 1H, ArH), 7.65
3374 (NeH), 2362 (eCN) and 1665 (C]O); 1H NMR (600 MHz,
CD3OD)
(d, J ¼ 7.8 Hz, 2H, ArH), 7.63 (s, 1H, ArH), 7.50 (d, J ¼ 7.2 Hz, 2H, ArH),
d
(ppm): 7.98 (d, J ¼ 7.8 Hz, 2H, ArH), 7.81 (d, J ¼ 8.4 Hz, 2H,
7.48 (s, 1H, ArH), 7.42 (d, J ¼ 8.4 Hz, 1H, ArH), 7.36 (t, J ¼ 7.8 Hz, 1H,
ArH), 7.66 (d, J ¼ 7.2 Hz, 1H, ArH), 7.58 (d, J ¼ 7.8 Hz, 2H, ArH), 7.54
(d, J ¼ 7.8 Hz, 2H, ArH), 7.34 (d, J ¼ 6.6 Hz, 1H, ArH), 7.26 (m, 2H,
ArH), 4.38 (s, 2H, CH2); MS (ESI) m/z: 587.3 ([M - H]þ). Anal. Calcd
for C27H15Cl2N7OS2: C, 55.11; H, 2.57; N, 16.66. Found: C, 55.17; H,
ArH), 4.42 (s, 2H, CH2); 13C NMR (150 MHz, DMSO-d6)
d (ppm):
166.1,135.9,134.2,132.1,131.5,131.4,129.3,129.2,129.0,128.9,128.7,
128.6, 127.7, 127.4, 127.3, 126.6, 44.5; MS (ESI) m/z: 656.3 ([M - H]þ).
Anal. Calcd. for C27H13Cl4N7OS2: C, 49.33; H, 1.99; N,14.91. Found: C,

164
P. Cui et al. / European Journal of Medicinal Chemistry 127 (2017) 159e165
49.25; H, 1.96; N, 14.84.
4.5. Computational modeling
The molecular structure of the compounds 7d was drawn with
Chemoffice Ultra 8.03 (CambridgeSoft, USA). Then the 3D structure
was optimized with Omega2 [38e40] on GSU cluster Orion [41].
Finally, the crystal structure of the SecA (PDBID: 2FSG [42]) was
docked with the compound 7d using Surflex-Dock [43]. We picked
the pocket in two methods: designating the ATPase ATP binding
site as the pocket using the co-crystalized ATP molecule and
following our previous procedures [31]. 20 outputs were generated
for docking score analysis.
4.2.11. 4-(2,6-Dichloro-phenyl)-2-{4-[3-(2,6-dichloro-phenyl) -
[1,2,4]triazolo [3,4-b] [1,3,4]thiadiazol-6-yl]-benzylsulfanyl} -6-oxo-
1,6-dihydro-pyrimidine-5-carbonitrile (7k)
Light yellow solid, Yield 65%; mp 230e233 ꢁC; IR (KBr, , cmꢀ1):
n
3360 (NeH), 2360 (eCN) and 1655 (C]O); 1H NMR (600 MHz,
DMSO-d6)
d
(ppm): 7.98 (d, J ¼ 7.8 Hz, 2H, ArH), 7.67 (d, J ¼ 7.2 Hz,
2H, ArH), 7.63 (d, J ¼ 7.8 Hz, 2H, ArH), 7.50 (d, J ¼ 7.8 Hz, 2H, ArH),
7.42 (d, J ¼ 8.4 Hz, 1H, ArH), 7.36 (t, J ¼ 7.8 Hz, 1H, ArH), 4.42 (s, 2H,
CH2); 13C NMR (150 MHz, DMSO-d6)
d (ppm): 166.0, 132.1, 131.8,
131.4, 131.0, 130.8, 130.5, 129.1, 129.0, 128.4, 127.8, 127.3, 52.3; MS
(ESI) m/z: 656.1 ([M - H]þ). Anal. Calcd. for C27H13Cl4N7OS2: C,
49.33; H, 1.99; N, 14.91. Found: C, 49.20; H, 1.98; N, 14.82.
Acknowledgments
This work was supported by the Post-Graduate's Innovation
Fund Project of Hebei Province, China (No. S2016003). Work in the
Wang lab (design and computational work) is supported by a grant
NIH (AI104168) from the United States. We gratefully acknowledge
the use of Orion that is supported by Georgia State University's
Research Solutions and the free academic licensing program of
Open eye. We gratefully acknowledge Dr Hui Tang in Hebei Uni-
versity for his helpful discussions on the antibacterial activities.
4.2.12. 4-Biphenyl-4-yl-2-{4-[3-(2,6-dichloro-phenyl)- [1,2,4]
triazolo [3,4-b] [1,3,4]thiadiazol-6-yl]-benzylsulfanyl}-6-oxo-1,6-
dihydro-pyrimidine-5-carbonitrile (7l)
Light yellow solid, Yield 65%; mp 238e241 ꢁC; IR (KBr, , cmꢀ1):
n
3364 (NeH), 2360 (eCN) and 1645 (C]O); 1H NMR (600 MHz,
DMSO-d6) (ppm): 7.95 (s, 2H, ArH), 7.94 (s, 2H, ArH), 7.77 (d,
d
J ¼ 8.4 Hz, 2H, ArH), 7.72 (d, J ¼ 7.8 Hz, 2H, ArH), 7.68 (d, J ¼ 8.4 Hz,
2H, ArH), 7.50 (s, 1H, ArH), 7.47 (d, J ¼ 3.6 Hz, 2H, ArH), 7.47 (s, 1H,
ArH), 7.40 (d, J ¼ 7.2 Hz,1H, ArH), 7.35 (t, J ¼ 7.8 Hz,1H, ArH), 4.54 (s,
Appendix A. Supplementary data
2H, CH2); 13C NMR (150 MHz, DMSO-d6)
d (ppm): 166.1,156.7,140.4,
139.4,138.8,134.9,134.1,133.9,132.1,131.5,131.4,128.9,128.8,128.6,
127.6, 127.3, 127.1, 126.7, 126.6, 126.5, 47.9; MS (ESI) m/z: 663.5
([M ꢀ H]þ). Anal. Calcd. for C33H19Cl2N7OS2: C, 59.64; H, 2.88; N,
14.75. Found: C, 59.56; H, 2.81; N, 14.69.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.12.053.
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