Design, synthesis, and biological evaluation of pyrimidine analogs as SecA inhibitors

Article abstract of DOI:10.1007/s00044-021-02717-6

SecA, a key component of the bacterial Sec-dependent secretion pathway, is an attractive target for the development of new antimicrobial agents. We have previously reported pyrimidine analogs as SecA inhibitors. Herein, we report an extension of the earlier work in the synthesis and evaluation of a series of 15 5-cyanothiouracil derivatives as SecA inhibitors. All the compounds have been evaluated for their inhibition of SecA ATPase (EcSecAN68) and for their antimicrobial activity against Escherichia coli NR698 (a leaky mutant) and Bacillus anthracis Sterne. Twelve compounds showed IC₅₀ of less than 6.3 μM when tested against EcSecAN68. In antimicrobial studies against E. coli NR698, six compounds showed MIC of <12.5 μM with three being less than 6.3 μM. Against B. anthracis Sterne, three compounds showed MIC of <6.3 μM.

Full text of DOI:10.1007/s00044-021-02717-6

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-021-02717-6
ORIGINAL RESEARCH
Design, synthesis, and biological evaluation of pyrimidine analogs
as SecA inhibitors
1,2
Jinshan Jin3,5 Arpana S. Chaudhary1,6 Phang C. Tai3 Binghe Wang1,4
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Fante Bamba
Received: 21 January 2021 / Accepted: 18 February 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021
Abstract
SecA, a key component of the bacterial Sec-dependent secretion pathway, is an attractive target for the development of new
antimicrobial agents. We have previously reported pyrimidine analogs as SecA inhibitors. Herein, we report an extension of
the earlier work in the synthesis and evaluation of a series of 15 5-cyanothiouracil derivatives as SecA inhibitors. All the
compounds have been evaluated for their inhibition of SecA ATPase (EcSecAN68) and for their antimicrobial activity
against Escherichia coli NR698 (a leaky mutant) and Bacillus anthracis Sterne. Twelve compounds showed IC50 of less than
6.3 μM when tested against EcSecAN68. In antimicrobial studies against E. coli NR698, six compounds showed MIC of
<12.5 μM with three being less than 6.3 μM. Against B. anthracis Sterne, three compounds showed MIC of <6.3 μM.
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Keywords SecA inhibitor Small molecule Antimicrobial Target Drug-resistant
Introduction
pathway to help protein translocation from the cytosol across
or into the cytoplasmic membrane [5–11]. Because SecA is a
conserved and essential protein in all bacteria and is absent
in humans, it is considered as a promising antibacterial drug
target. At present, small organic molecules that can inhibit
SecA mainly include Rose Bengal [12], bisthiouracil [13],
bistriazole [14] and their derivatives [15], thiazolo[4,5-d]
pyrimidine derivatives [16], and others [17, 18].
As an extension of our earlier work [15, 19], we are
working on optimizing existing SecA inhibitors of the sub-
stituted pyrimidine scaffold by exploring the chemistry space
at the 2 and 4 positions of the pyrimidine core [13]. Below, we
describe the results and implications in guiding future work in
this area.
Infectious diseases caused by bacterial pathogens have
become a major public health problem in recent years due to
the widespread occurrence of drug resistance. Therefore,
there is an urgent need to develop new antimicrobial agents,
especially those with a new drug target and/or with the
ability to overcome drug resistance [1]. Along this line, we
are interested in targeting SecA, which is a critical protein
secretion machinery essential for bacterial survival [2–4].
SecA is a key component of the bacterial protein secretion
(Sec) pathways. SecA ATPase is one of the essential com-
ponents in the Sec machinery, which provides a major
Supplementary information The online version contains
supplementary material available at https://doi.org/10.1007/s00044-
021-02717-6.
* Phang C. Tai
3
Departments of Biology, Georgia State University, Atlanta, GA
30303, USA
biopct@gsu.edu
* Binghe Wang
4
Center for Diagnostics and Therapeutics, Georgia State University,
Atlanta, GA 30303, USA
wang@gsu.edu
5
Present address: US Food and Drug Administration, National
Center of Toxicological Research, 3900 NCTR Road,
Jefferson, AR 72079, USA
1
Departments of Chemistry, Georgia State University, Atlanta, GA
30303, USA
2
Laboratoire de Chimie Organique et des Substances Naturelles,
Université Félix Houphouët-Boigny, 22Bp 582, Abidjan, Côte
d’Ivoire
6
Present address: Bayer Corporation, 800N. Lindbergh Blvd,
St. Louis, MO 63141, USA

Medicinal Chemistry Research
Results and discussion
provide amide 1f by following a published procedure
[24]. The reaction of the appropriate aromatic aldehydes,
cyano ethyl acetate and thiourea with piperidine as cata-
lyst in absolute ethanol at reflux temperature overnight
afforded thiouracil 2a–f. The target compounds 3a–o were
obtained by the S-benzylation in the presence of potas-
sium carbonate in acetonitrile. Overall, 15 compounds
have been prepared in overall yields ranging from 47–98
using readily available starting materials.
Chemistry
Earlier work has identified the compound in Fig. 1 as a lead
for further optimization [15, 19].
To improve the potency, we focused our attention at the
linker between the biphenyl rings and the para position of
the benzylthio group. Specifically, the designed com-
pounds were synthesized by introducing (a) linkers such
as thioethers (–S–, –CH2–S–), an inverted alkoxy
(–O–CH2–), an alkenyl group (–CH=CH–), an alkynyl
(–C≡C–) group, and an amido group (-CH2-NH-CO-)
between the two phenyl rings and (b) substituents such as
–N3, –COOMe, -CF3 at the para position of the ben-
zylthioether moiety. Thus, a series of pyrimidine deriva-
tives 3a–o were synthesized. The synthetic route is shown
in Scheme 1. Compounds 1a, b were prepared by
nucleophilic aromatic substitution reaction of 4-
nitrobenzaldehyde [20] or 4-fluorobenzaldehyde [21]
using the appropriate aryl thiols under basic conditions.
Compound 1c was obtained by following a published
procedure [20] in Scheme 2. Palladium catalyzed
Mizoroki-Hech reaction of 4-bromobenzaldehyde with
styrene yielded 1d [22]. Compound 1e was prepared by
palladium catalyzed Sonogoshira cross coupling of 4-
bromobenzaldehyde with phenylacetylene [23]. Then 4-
formylbenzoic acid was coupled with benzylamine using
isobutyl chloroformate in the presence of triethylamine to
Biological evaluation
The activities of all newly synthesized compounds 3a–o
were first screened using a truncated version of E. coli
SecA, EcSecAN68, at 6.25 μM [15]. As it can be seen in
Fig. 2, most of these 15 new compounds showed more
than 50% inhibition of the SecA ATPase activity at this
concentration, suggesting potent inhibitory activity. Some
of the compounds seemed to be more potent than the lead
compound, SCA168, and showed more than 90% inhibi-
tion at 6.25 μM such as SCA225, 227, 230, 232, 233, 239,
245, 260, and 262. The overall results from the SecA
inhibition studies seem to suggest that the “linker” part
can accommodate thioethers (SCA225, 230, 239, and
SCA260–262), an inverted oxo ether (SCA232, 238, 240),
an alkenyl linker (SCA227, 233), and alkynyl linker
(SCA245) as well as variations in the length by one
methylene group (1–2 atoms) among those with a thioe-
ther linker. Among all these variations, it is especially
interesting to see the accommodation of the linearization
of the linker with an alkynyl group (SCA245). It also
seems that the para-position of the thiobenzyl group of the
4-position of the pyrimidine ring can tolerate various
groups. From this limited set of compounds, it is also clear
that a three-atom linker in the form of an amido group is
not well-tolerated.
All compounds were also evaluated for their anti-
microbial activity against a “leaky” outer membrane mutant
of Gram-negative E. coli NR698 [25]. Among them, four
compounds showed minimum inhibitory concentration
(MIC) at about 12.5 μM (SCA225, 232, 240, 261) and two
compounds showed MIC at about 6.3 μM (SCA260, 262)
against E. coli NR698 (Table 1). Compounds were also
evaluated for their antimicrobial activity against Gram
positive B. anthracis Sterne (Table 2). Three of them
showed MIC at about 6.3 μM (SCA225, 227, 232) against
B. anthracis Sterne, which are comparable to some of our
best compounds in this class [15]. With the limited data set,
it seems that the potency against B. anthracis is higher than
that of E. coli. Such results are understandable because E.
coli is Gram-negative strain. Even with a compromised
outer membrane, it still has an outer membrane, which
presents a permeability barrier [26].
Fig. 1 Lead compound for the synthesis of novel SecA inhibitors

Medicinal Chemistry Research
Scheme 1 Synthesis of compound 3a–o
Scheme 2 Synthesis of compound 1c–e
Overall, the results provide very useful information for
those who might be interested in SecA inhibitor design for
both improved potency and for avoiding the chemical space
that would not be productive.
Conclusion
We have described the design, syntheses, and biological
evaluation of a series of 15 small-molecule SecA inhibitors

Medicinal Chemistry Research
with μM inhibition. The results provide information on
tolerable structural features of the linker part between the
two phenyl rings at the 2-position and the para-substitution
of the benzyl group at the 4-position of the pyrimidine ring.
Upon completion, the reaction mixture was cooled to ambient
temperature and the solvent removed in vacuo. The dried
residue was washed by H2O (pH = 9–10, 20 ml × 2) and brine
(15 ml × 2) followed by product extraction in ethyl acetate
(20 ml). The solvent was evaporated in vacuo to obtain a
crude product, which was purified using silica gel column
chromatography.
Experimental
All chemical reagents and solvents used were of reagent
grade or purified using standard methods. TLC analyses
were conducted on silica gel plates (Sorbent Silica G
UV254). Column chromatography was carried out on flash
silica gel (Sorbent 230–400 mesh). NMR spectra were
2-Mercapto-6-oxo-4-(4-(phenylthio)phenyl)-1,6-dihydropyr-
imidine-5-carbonitrile (2a) Yield: 58%
1H NMR (DMSO-d6): δ 13.26 (bs, 1H), 12.91 (s, 1H),
7.63 (d, J = 8.4 Hz, 2H), 7.50 (m, 5H), 7.31 (d, J = 8.4 Hz,
2H); 13C NMR (DMSO-d6): δ 177.6, 161.5, 159.5, 142.3,
133.8, 132.0, 131.9, 131.2, 130.8, 130.5, 130.1, 130.1,
129.5, 128.5, 127.9, 115.8, 90.1 ppm. HRMS (ESI-TOF)
(m/z): Calcd. for C17H10N3OS2, [M–H+]: 336.0260; found:
336.0263.
1
recorded at H (400 MHz) and 13C (100 MHz) on a Bruker
instrument. Coupling constants (J) and chemical shifts (δ)
are given in hertz and ppm, respectively, using TMS (1H
NMR) and solvents (13C NMR) as internal standards.
General procedure for the synthesis of (2a–f)
4-(4-(Benzylthio)phenyl)-2-mercapto-6-oxo-1,6-dihydropyri-
midine-5-carbonitrile (2b) Yield: 45%
Our previously published [15] procedure was followed.
Briefly, to a solution of ethanol (25 ml) and appropriate
aldehyde (RCHO, 5 mmol) was added ethyl cyanoacetate
(0.5 ml, 5 mmol), thiourea (0.38 g, 5 mmol) and piperidine
(1.0 ml, 10 mmol). The mixture was heated at reflux over-
night and then cooled to room temperature. The precipitate
was dissolved in 0.5 M NaOH (20 ml) and washed with
ethyl acetate (15 ml × 3). Then the aqueous solution was
acidified to pH ~2 by slow addition of 1 M HCl. This
caused the product to precipitate, which was then filtered
using vacuum filtration.
1H NMR (DMSO-d6): δ 13.81 (s, 1H), 13.17 (bs, 1H),
7.61 (d, J = 8.8 Hz, 2H), 7.47 (d, J = 8.4 Hz, 2H), 7.44 (d,
J = 7.2 Hz, 2H), 7.33 (t, J = 7.6 Hz, 2H), 7.25 (m, 1H), 4.37
(s, 2H); 13C NMR (DMSO-d6): δ 178.1, 162.1, 159.9,
142.4, 137.3, 129.7, 129.3, 129.0, 128.1, 127.7, 126.6,
116.2, 89.5, 35.8. HRMS (ESI-TOF) (m/z): Calcd. for
C18H12N3OS2, [M-H+]: 350.0416; found: 350.0420.
2-Mercapto-6-oxo-4-(4-(phenoxymethyl)phenyl)-1,6-dihy-
dropyrimidine-5-carbonitrile (2c) Yield: 36%
1H NMR (DMSO-d6): δ 13.31 (bs, 1H), 13.14 (s, 1H),
7.71 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 8.0 Hz, 2H), 7.31 (t,
J = 7.6 Hz, 2H), 7.04 (d, J = 8.0 Hz, 2H), 6.96 (t, J =
7.2 Hz, 1H), 4.37 (s, 2H); 13C NMR (DMSO-d6): δ 176.8,
161.3, 159.2, 158.6, 142.1, 130.0, 129.5, 127.8, 121.5,
115.4, 91.1, 68.9. HRMS (ESI-TOF) (m/z): Calcd. for
C18H12N3O2S, [M-H+]: 334.0645; found: 334.0645.
General procedure for the synthesis of (3a–o)
To a solution of 2-mercapto-6-oxo-4-(4-(phenylthio)phenyl)-
1,6-dihydropyrimidine-5-carbonitrile derivatives (1.36 mmol)
in CH3CN (10 ml), K2CO3 (6.79 mmol) was added and the
resulting mixture was stirred for 10–15 min. To this was added
the appropriate (bromomethyl)phenyl derivatives (1.22 mmol)
and the reaction was stirred at room temperature for 16–18 h.
(E)-2-Mercapto-6-oxo-4-(4-styrylphenyl)-1,6-dihydropyrimi-
dine-5-carbonitrile (2d) Yield: 56%
1H NMR (DMSO-d6): δ 13.32 (bs, 1H), 12.93 (s, 1H),
7.78 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 8.4 Hz, 2H), 7.66 (d,
Table 2 SecA inhibitors with MIC of <50 μM against B.
anthracis Sterne
SCA225 SCA227 SCA230 SCA232 SCA168
MIC (μM) 6.25
6.25
25
6.25
6.25
Fig. 2 Screening for inhibition of the ATPase activity of EcSecAN68
Table 1 SecA inhibitors with
SCA225 SCA232 SCA238 SCA239 SCA240 SCA260 SCA261 SCA262 SCA168
12.5 50 50 12.5 6.25 12.5 6.25 12.5
MIC of <50 μM against E.
coli NR698
MIC (μM) 12.5

Medicinal Chemistry Research
J = 7.2 Hz, 2H), 7.37–7.48 (m, 4H), 7.33 (d, J = 6.0 Hz,
1H); 13C NMR (DMSO-d6): δ 177.5, 161.7, 159.5, 141.1,
137.0, 131.5, 129.7, 129.4, 129.2, 128.9, 128.7, 127.7,
127.3, 126.6, 115.8, 90.0. HRMS (ESI-TOF) (m/z): Calcd.
for C19H12N3OS, [M-H+]: 330.0696; found: 330.0698.
118.0, 91.4, 55.3, 33.8. HRMS (ESI-TOF) (m/z): Calcd. for
C25H15F3N3OS2, [M-H+]: 494.0614; found: 494.0598.
2-((4-Azidobenzyl)thio)-4-(4-(benzylthio)phenyl)-6-oxo-1,6-
dihydropyrimidine-5-carbonitrile (3d) Yield: 40%
1H NMR (DMSO-d6): δ 7.90 (d, J = 8.0 Hz, 2H), 7.50 (d,
J = 8.4 Hz, 2H), 7.44 (d, J = 7.6 Hz, 4H), 7.33 (t, J =
8.0 Hz, 2H), 7.26 (m, 1H), 7.06 (d, J = 8.4 Hz, 2H), 4.52 (s,
2H), 4.39 (s, 2H); 13C NMR (DMSO-d6): δ 166.6, 165.7,
161.5, 144.8, 142.8, 139.0, 137.2, 133.9, 132.1, 131.1,
129.3, 128.9, 127.7, 126.8, 119.6, 116.4, 92.8, 35.7, 34.1.
HRMS (ESI-TOF) (m/z): Calcd. for C25H17N6OS2, [M-H+]:
481.0905; found: 481.0911.
2-Mercapto-6-oxo-4-(4-(phenylethynyl)phenyl)-1,6-dihydro-
pyrimidine-5-carbonitrile (2e) [15] Yield: 58%
1H NMR (DMSO-d6): δ 10.03 (s, 1H), 7.87 (d, J =
8.0 Hz, 2H), 7.69 (d, J = 8.0 Hz, 2H), 7.57 (t, J = 8.0 Hz,
2H), 7.39 (t, J = 8.0 Hz, 2H); 13C NMR (DMSO-d6): δ
171.4, 135.4, 132.1, 131.8, 129.6, 129.0, 128.5, 122.5,
93.4, 88.5. HRMS (ESI-TOF) (m/z): Calcd. for
C19H10N3OS, [MH-]: 328.0545; found: 328.0541.
Methyl 4-(((4-(4-(benzylthio)phenyl)-5-cyano-6-oxo-1,6-dihydro-
pyrimidin-2-yl)thio)methyl) benzoperoxoate (3e) Yield: 68%
1H NMR (DMSO-d6): δ 7.88 (d, J = 8.0 Hz, 2H), 7.69 (d,
J = 8.0 Hz, 2H), 7.53 (d, J = 8.0 Hz, 2H), 7.42 (t, J =
6.4 Hz, 4H), 7.31 (t, J = 7.2 Hz, 2H), 7.24 (m, 1H), 4.34 (s,
2H), 4.32 (s, 2H); 13C NMR (DMSO-d6): δ 171.6, 170.8,
166.6, 166.5, 145.6, 139.3, 137.6, 135.0, 129.6, 129.3,
129.2, 129.0, 128.9, 128.4, 127.6, 127.2, 127.0, 120.5,
89.1, 52.5, 36.3, 33.8. HRMS (ESI-TOF) (m/z): Calcd. for
C27H20N3O3S2, [M-H+]: 498.0946; found: 498.0953.
N-Benzyl-4-(5-cyano-2-mercapto-6-oxo-1,6-dihydropyrimi-
din-4-yl)benzamide (2f) Yield: 58%
1H NMR (DMSO-d6): δ 13.41 (s, 1H), 13.20 (s, 1H), 9.26
(t, J = 6.0 Hz, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.78 (d, J =
8.0 Hz, 2H), 7.34 (d, J = 4.4 Hz, 4H), 7.28 (m, 1H), 4.51 (d,
J = 6.0 Hz, 2H); 13C NMR (DMSO-d6): 176.6, 165.8, 160.7,
158.8, 139.8, 137.8, 132.1, 129.5, 129.4, 128.7, 127.8, 127.6,
127.2, 115.0, 91.6, 43.2. HRMS (ESI-TOF) (m/z): Calcd. for
C19H13N4O2S, [M-H+]: 361.0754; found: 361.0756.
2-((4-Azidobenzyl)thio)-6-oxo-4-(4-(phenylthio)phenyl)-1,6-
dihydropyrimidine-5-carbonitrile (3a) Yield: 90%
4-(4-(Benzylthio)phenyl)-6-oxo-2-((4-(trifluoromethyl)benzyl)
thio)-1,6-dihydropyrimidine-5-carbonitrile (3 f) Yield: 59%
1H NMR (DMSO-d6): δ 7.83 (d, J = 8.4 Hz, 2H), 7.68 (d,
J = 8.4 Hz, 2H), 7.63 (d, J = 8.0 Hz, 2H), 7.48 (d, J =
8.0 Hz, 4H), 7.44 (d, J = 7.2 Hz, 2H), 7.32 (t, J = 7.6 Hz,
2H), 7.25 (m, 1H), 4.59 (s, 2H), 4.38 (s, 2H); 13C NMR
(DMSO-d6): δ 166.7, 165.8, 142.5, 137.2, 132.2, 130.1,
129.6, 129.3, 128.9, 127.7, 126.8, 125.8, 125.7, 117.02,
92.9, 35.8, 33.9. HRMS (ESI-TOF) (m/z): Calcd. for
C26H17F3N3OS2, [M-H+]: 508.076; found: 508.0773.
1H NMR (DMSO-d6): δ 7.83 (d, J = 8.0 Hz, 2H), 7.49
(m, 3H), 7.43 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H),
7.04 (d, J = 8.0 Hz, 2H), 4.51 (s, 2H); 13C NMR (DMSO-
d6): δ 169.2, 166.3, 166.6, 166.4, 140.4, 138.6, 135.2,
134.8, 133.2, 132.8, 131.0, 130.3, 129.8, 129.0, 128.6,
119.5, 118.4, 91.1, 33.9. HRMS (ESI-TOF) (m/z): Calcd.
for C24H15N6OS2, [M-H+]: 467.0749; found: 467.0751.
Methyl4-(((5-cyano-6-oxo-4-(4-(phenylthio)phenyl)-1,6-dihy-
dropyrimidin-2-yl)thio)methyl)benzoate (3b) Yield: 97%
1H NMR (DMSO-d6): δ 7.87 (d, J = 8.4 Hz, 2H), 7.77 (d,
J = 8.0 Hz, 2H), 7.54 (d, J = 8.0 Hz, 2H), 7.41–7.49 (m,
2-((4-Azidobenzyl)thio)-6-oxo-4-(4-(phenoxymethyl)phe-
nyl)-1,6-dihydropyrimidine-5-carbonitrile (3 g) Yield: 66%
1H NMR (DMSO-d6): δ 7.96 (d, J = 8.0 Hz, 2H), 7.65 (d,
J = 7.6 Hz, 2H), 7.45 (d, J = 8.0 Hz, 4H), 7.31 (t, J =
7.6 Hz, 2H), 7.05 (t, J = 8.8 Hz, 4H), 6.96 (t, J = 6.8 Hz,
1H), 5.22 (s, 2H), 4.53 (s, 2H); 13C NMR (DMSO-d6): δ
167.4, 166.01, 161.6, 158.5, 141.6, 139.1, 135.0, 133.8,
131.1, 130.0, 129.4, 127.9, 127.8, 121.3, 119.6, 116.3,
115.2, 93.6, 68.8, 34.1. HRMS (ESI-TOF) (m/z): Calcd. for
C25H17N6O2S, [M-H+]: 465.1134; found: 465.1149.
5H), 7.31 (d, J = 8.4 Hz, 2H), 4.42 (s, 2H), 3.83 (s, 3H); 13
C
NMR (DMSO-d6): δ 170.0, 166.6, 166.4, 144.6, 139.7,
135.2, 133.0, 130.3, 129.7, 129.6, 129.6, 128.9, 128.8,
128.6, 119.0, 90.5, 52.5, 34.0. HRMS (ESI-TOF) (m/z):
Calcd. for C26H18N3O3S2, [M-H+]: 484.0795; found:
484.0774.
6-Oxo-4-(4-(phenylthio)phenyl)-2-((4-(trifluoromethyl)benzyl)
thio)-1,6-dihydropyrimidine-5-carbonitrile (3c) Yield: 90%
1H NMR (DMSO-d6): δ 7.79 (d, J = 8.4 Hz, 2H), 7.64
(qd, J = 8.4 Hz, 4H), 7.43–7.51 (m, 5H), 7.31(d, J =
8.0 Hz, 2H), 4.49 (s, 2H); 13C NMR (DMSO-d6): δ 168.6,
166.7, 166.0, 143.4, 140.7, 134.5, 133.3, 130.3, 130.0,
129.8, 129.1, 128.5, 128.2, 127.9, 126.0, 125.6, 123.3,
Methyl-4-(((5-cyano-6-oxo-4-(4-(phenoxymethyl)phenyl)-1,6-
dihydropyrimidin-2-yl)thio)methyl)benzoate (3h) Yield: 68%
1H NMR (DMSO-d6): δ 7.90 (d, J = 8.0 Hz, 4H), 7.62 (d,
J = 8.0 Hz, 2H), 7.57 (d, J = 8.0 Hz, 2H), 7.31 (t, J =
7.6 Hz, 2H), 7.04 (d, J = 7.6 Hz, 2H), 6.96 (t, J = 7.2 Hz,
1H), 5.02 (s, 2H), 4.57 (s, 2H), 3.84 (s, 3H); 13C NMR

Medicinal Chemistry Research
(DMSO-d6): δ 171.4, 167.5, 166.4, 158.6, 143.1, 141.5,
135.2, 130.0, 129.7, 129.3, 127.9, 121.3, 115.2, 108.6,
93.4, 68.9, 52.6, 34.2. HRMS (ESI-TOF) (m/z): Calcd. for
C27H20N3O4S, [M-H+]: 482.1175; found: 482.1175.
(DMSO-d6): δ 167.1, 166.7, 166.0, 162.0, 139.9, 139.0, 138.2,
137.3, 134.1, 131.1, 129.2, 128.7, 127.9, 127.2, 119.6, 116.3,
93.9, 43.2, 34.1. HRMS (ESI-TOF) (m/z): Calcd. for
C26H18N7O2S, [M-H+]: 492.1248; found: 492.1232.
6-Oxo-4-(4-(phenoxymethyl)phenyl)-2-((4-(trifluoromethyl)ben-
zyl)thio)-1,6-dihydropyrimidine-5-carbonitrile (3i) Yield: 65%
1H NMR (DMSO-d6): δ 7.90 (d, J = 8.0 Hz, 2H),
7.62–7.69 (m, 6H), 7.31 (t, J = 7.6 Hz, 2H), 7.04 (d, J =
8.0 Hz, 1H), 6.95 (t, J = 7.2 Hz, 1H), 5.22 (s, 2H), 4.61 (s,
2H); 13C NMR (DMSO-d6): δ 168.6, 167.4, 165.9, 158.5,
142.4, 141.6, 135.0, 130.2, 130.0, 129.3, 127.9, 125.8,
125.7, 121.3, 116.4, 115.2, 93.7, 68.8, 33.9. HRMS (ESI-
TOF) (m/z): Calcd. for C26H17F3N3O2S, [M-H+]: 492.0994;
found: 492.0989.
Methyl 4-(((4-(4-(benzylcarbamoyl)phenyl)-5-cyano-6-oxo-1,6-
dihydropyrimidin-2-yl)thio) methyl)benzoate (3n) Yield: 63%
1H NMR (DMSO-d6): δ 9.24 (t, J = 6.0 Hz 1H), 7.98 (d,
J = 8.0 Hz, 2H), 7.88 (dd, J = 8.0 Hz, 4H), 7.55 (d, J =
8.0 Hz, 2H), 7.34 (d, J = 4.4 Hz, 4H), 7.25 (m, 1H), 4.51 (d,
J = 6.0 Hz, 2H), 4.44 (s, 2H); 13C NMR (DMSO-d6): δ
170.3, 168.0, 166.9, 166.5, 166.1, 144.7, 139.9, 139.7,
136.2, 129.6, 128,7, 128.6, 127.6, 127.2, 118.9, 91.0, 52.5,
43.1, 34.0. HRMS (ESI-TOF) (m/z): Calcd. for
C28H21N4O4S, [M-H+]: 509.1289; found: 509.1271.
1H NMR (DMSO-d6): δ 8.00 (d, J = 8.0 Hz, 2H), 7.81 (d,
J = 8.4 Hz, 2H), 7.66 (d, J = 7.6 Hz, 2H), 7.48 (d, J =
8.4 Hz, 2H), 7.40 (m, 3H), 7.33 (d, J = 6.4 Hz, 2H), 7.08 (d,
N-Benzyl-4-(5-cyano-6-oxo-2-((4-(trifluoromethyl)benzyl)thio)-
1,6-dihydropyrimidin-4-yl)benzamide (3o) Yield: 68%
1H NMR (DMSO-d6): δ 9.21 (t, J = 6.0 Hz 1H), 8.02 (d, J
= 8.0 Hz, 2H), 7.92 (d, J = 8.4 Hz, 2H), 7.66 (qd, J =
8.0 Hz, 4H), 7.34 (d, J = 4.4 Hz, 4H), 7.26 (m, 1H), 4.58 (s,
2H), 4.52 (d, J = 6.0 Hz, 2H); 13C NMR (DMSO-d6): δ
167.2, 167.1, 166.0, 163.1, 142.8, 139.9, 138.5, 137.1,
130.2, 129.1, 128.7, 128.4, 128,1, 127.8, 127.2, 126.0,
125.7, 123.3, 116.8, 93.5, 43.2, 33.9. HRMS (ESI-TOF)
(m/z): Calcd. for C27H18F3N4O2S, [M-H+]: 519.1097; found:
519.1091.
J = 5.2 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 5.55 (s, 2H); 13
C
NMR (DMSO-d6): 167.6, 166.9, 165.9, 141.1, 139.1,
137.1, 134.0, 131.5, 131.1, 129.7, 129.2, 128.9, 128.6,
128.3, 127.8, 127.3, 126.9, 119.7, 93.0, 34.1. HRMS (ESI-
TOF) (m/z): Calcd. for C26H17N6OS, [M-H+]: 461.1185;
found: 461.1179.
(E)-Methyl 4-(((5-cyano-6-oxo-4-(4-styrylphenyl)-1,6-dihy-
dropyrimidin-2-yl)thio)methyl) benzoate (3k) Yield: 34%
1H NMR (DMSO-d6): δ 7.94 (d, J = 8.4 Hz, 2H), 7.91 (d,
J = 7.6 Hz, 2H), 7.79 (d, J = 7.6 Hz, 2H), 7.66 (d, J =
7.6 Hz, 2H), 7.59 (d, J = 7.6 Hz, 2H), 7.42 (d, J = 6.4 Hz,
2H), 7.39 (d, J = 5.2 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 5.61
(s, 2H), 3.84 (s, 3H); 13C NMR (DMSO-d6): δ 167.0, 166.5,
165.9, 162.1, 143.2, 141.1, 137.1, 134.4, 131.4, 129.7,
129.2, 129.0, 128.7, 127.7, 127.2, 126.9, 116.7, 93.1, 52.6,
34.1. HRMS (ESI-TOF) (m/z): Calcd. for C28H20N3O3S,
[M-H+]: 478.1225; found: 478.1240.
ATPase assays
Inhibition on ATPase activity of EcSecAN68 was deter-
mined by malachite green colorimetric assay as previously
described [15]. IC50 is defined as the concentration of the
compound that inhibits 50% of ATPase activity.
Bacteriostatic effects
6-Oxo-4-(4-(phenylethynyl)phenyl)-2-((4-(trifluoromethyl)ben-
zyl)thio)-1,6-dihydropyrimidine-5-carbonitrile (3l) Yield: 51%
1H NMR (DMSO-d6): δ 7.93 (d, J = 8.0 Hz, 2H), 7.64–7.75
(m, 6H), 7.65 (m, 2H), 7.46(m, 3H), 4.60 (s, 2H); 13C NMR
(DMSO-d6): δ 166.6, 162.5, 142.6, 135.6, 132.0, 131.9,
130.2, 129.6, 129.4, 129.3, 125.9, 125.8, 122.3, 116.5, 93.7,
92.3, 89.1, 33.9. HRMS (ESI-TOF) (m/z): Calcd. for
C27H16F3N3OS, [M-H+]: 486.0888; found: 486.0884.
Bacteriostatic effects were evaluated at 37 °C in 96-well
microtitier plates as previously described [15]. Minimum
inhibitory concentration (MIC) is the lowest concentration
of compounds at which bacterial cells were not able to grow
at tested condition.
Acknowledgements FB was a visiting scholar at GSU when con-
ducting the lab research with partial financial support from the
Islamic Development Bank (IDB) under a merit scholarship pro-
gram. We also acknowledge the partial financial support to PCT and
BW (AI104168) by the National Institutes of Health. JJ and ASC
were supported by the Molecular Basis of Diseases Fellowship of
Georgia State University.
4-(2-((4-Azidobenzyl)thio)-5-cyano-6-oxo-1,6-dihydropyri-
midin-4-yl)-N-benzylbenzamide (3m) Yield: 55%
1H NMR (DMSO-d6): δ 9.24 (t, J = 6.0 Hz 1H), 8.04 (d,
J = 8.4 Hz, 2H), 7.98 (d, J = 8.4 Hz, 2H), 7.45 (d, J = 8.4 Hz,
2H), 7.34 (d, J = 4.0 Hz, 4H), 7.26 (m, 1H), 7.12 (t, J =
7.6 Hz, 1H), 7.07 (d, J = 8.4 Hz, 2H), 4.51 (m, 4H); 13C NMR
Compliance with ethical standards
Conflict of interest The authors declare no competing interests.

Medicinal Chemistry Research
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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