Synthesis and biological evaluation of novel 4-oxo-5-cyano thiouracil derivatives as SecA inhibitors

Article abstract of DOI:10.1515/hc-2020-0100

The continuous emergence of drug-resistant strains of bacteria poses an urgent risk to human health and dictates the need for new antimicrobials. Along this line, we have been working on developing inhibitors of SecA, a key component of the bacterial Sec-dependent secretion machinery. Herein, we describe the synthesis and antimicrobial evaluation of 6-oxo-1,6-dihydropyrimidine- 5-carbonitrile derivatives as potential SecA inhibitors.

Full text of DOI:10.1515/hc-2020-0100

Heterocycl. Commun. 2020; 26: 76–83
Research Article
Open Access
Fante Bamba, Jinshan Jin, Phang C. Tai* and Binghe Wang*
Synthesis and biological evaluation of novel
4-oxo-5-cyano thiouracil derivatives as SecA
inhibitors
https://doi.org/10.1515/hc-2020-0100
Received November 06, 2019; accepted March 11, 2020.
ATPase-driven “motor,” which couples the hydrolysis of
ATP to the stepwise translocation of preproteins across
Abstract: The continuous emergence of drug-resistant the bacterial cytoplasmic membrane [5-8]. Because SecA
strains of bacteria poses an urgent risk to human health is a conserved and essential protein in all bacteria and is
and dictates the need for new antimicrobials. Along this absent in humans, it is considered as a promising antibac-
line, we have been working on developing inhibitors of terial drug target. Various SecA inhibitors (Figure 1) have
SecA, a key component of the bacterial Sec-dependent been described in the literature [9]. Prior to our recent
secretion machinery. Herein, we describe the synthesis efforts, sodium azide was known as a SecA inhibitor and
and antimicrobial evaluation of 6-oxo-1,6-dihydropyrimi- was reported to possess antibacterial activity with IC50 of
dine-5-carbonitrile derivatives as potential SecA inhibitors. 1mM. The first natural product inhibitor of SecA, CJ-21058
[10] isolated from an unidentified fungus, showed anti-
Keywords: SecA inhibitor, small molecule, antimicrobial, bacterial activity against Gram-positive bacteria by inhi-
target, drug-resistant
biting the SecA translocation ATPase activity. Additional
examples include pannomycin [11], thiazolo[4,5-d]pyri-
midine derivatives [12], and others [13, 14]. During our
studies on developing small molecule SecA inhibitors, we
reported a structure-based virtual screening approach for
Introduction
Bacterial infection remains a serious threat to human the discovery of small-molecule inhibitors of the intrin-
health, largely because of the continuous emergence of sic ATPase activity of SecA [15]. Later on, we described
drug-resistance strains of bacteria [1, 2]. To address this optimization work [16] and inhibitors of different struc-
concern, both the World Health Organization (WHO) [3] tural scaffolds [17, 18]. Among all the efforts, we reported
and the US Center for Disease Control (CDC) [4] issued calls 4-oxo-5-cyano thiouracil derivatives as SecA inhibitors
for urgent actions. Therefore, there has been much effort [19]. Herein, we describe our efforts in optimizing a hit
in search of new antimicrobials. Especially desirable are (Figure 1), a 4-oxo-5-cyano thiouracil, and evaluation of
efforts toward identifying and inhibiting new targets and/ new analogs for antibacterial activities. The work is based
or new mechanisms of actions. Along this line, we have on the general structural scaffold of the lead with the
been working on developing SecA inhibitors as a way to aim of searching for the surrounding chemical space [16].
achieve antimicrobial activities. SecA is a key component Below, we describe the results and implications in guiding
of the bacterial protein secretion (Sec) pathways. It is an future work in this area.
*Corresponding authors: Phang C. Tai and Binghe Wang,
Results and discussion
Chemistry
Departments of Chemistry and Center for Diagnostics and
Therapeutics, Georgia State University, Atlanta, Georgia 30303,
USA, e-mail: wang@gsu.edu
Fante Bamba, Departments of Chemistry, Georgia State University,
Atlanta, Georgia 30303, USA; Laboratoire de Chimie Organique
et des Substances Naturelles, Université Félix Houphouët-Boigny,
22 Bp 582 Abidjan 22, COTE D‘IVOIRE
Jinshan Jin, Departments of Biology, Georgia State University,
Atlanta, Georgia 30303, USA
Based on our earlier work [19], the compound in Figure 1
was the lead compound for further exploration of the
chemical space that could inhibit SecA. In this respect, we
focused our attention on the para position of the phenyl
Open Access. © 2020 Bamba et al., published by De Gruyter.ꢀ
Attribution alone 4.0 License.
ꢀThis work is licensed under the Creative Commons

F. Bamba et al.: Design of novel SecA inhibitorsꢁ
ꢁ77
group, para position of the benzylthio group and the
linker between the biphenyl rings. Specifically, we desig-
ned compounds with various substituents such as F-, Me-,
MeO- on the phenyl group at position 4; linkers such as
-CH2O-, -O-, or -HN-CO- between the two phenyl rings, and
substituents such as -N3, -COOMe, or -CF3 at the 4-position
of the benzylthioether moiety. Such variations led to com-
pounds 3a-w.
The synthetic route of the compounds 3a-w is out-
lined in Scheme 1. Compounds 1a-e were prepared by
O-benzylation of 4-hydroxybenzaldehyde using the appro-
priate aryl halides under basic conditions [20]. The cor-
responding 4-formylbenzoic acid was coupled with the
Figure 1ꢂLead compound for the synthesis of novel SecA inhibitors.
Scheme 1ꢂSyntheses of compounds 3a-w.

ꢁ
78ꢁ
F. Bamba et al.: Design of novel SecA inhibitors
respective phenyl/aryl amines using isobutyl chlorofor- UV254). Column chromatography was carried out on flash
mate in the presence of triethylamine to provide amide silica gel (Sorbent 230–400 mesh). NMR spectra were
1
13
of type 1g-h by published procedure [21]. Following the recorded at H (400 MHz) and C (100 MHz) on a Bruker
methods for obtaining the core scaffold previously [16], instrument. Coupling constants (J) and chemical shifts
compounds 2a-I were obtained by condensation of an (δ) are given in hertz and ppm respectively, using TMS (1H
appropriate aldehyde with ethyl cyanoacetate and thiou- NMR) and solvents (13C NMR) as internal standards.
rea in the presence of piperidine. This was followed by the
General procedure for the synthesis of (2a-i): Our
S-benzylation in the presence of potassium carbonate in previously published [24] procedure was followed. Briefly,
acetonitrile to obtain compounds 3a-w.
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 under reflux overnight
and then cooled to room temperature. The precipitate was
Biological evaluation
The activities of the synthesized compounds were evalu- dissolved in 0.5 M NaOH (20 mL) and washed with ethyl
ated in a MIC assay using established procedures using acetate (15 ml ×3). Then the aqueous solution was acidi-
a mutant of Escherichia. coli with a compromised outer fied to pH ~ 2 by slow addition of 1 M HCl. This caused
membrane [19]. It was found that most of the compounds the product to precipitate, which was then filtered using
were not active at concentrations as high as 250 µM. Three vacuum filtration.
compounds showed MIC at about 20 µM (3i, l, w), and
one at about 8 µM (3m), which is comparable to some of To a solution of 4-(4-(benzyloxy)phenyl)-2-mercapto-
our best compounds in this class [22]. In addition, select 6-oxo-1,6-dihydropyrimidine-5-carbonitrile derivatives
General procedure for the synthesis of (3a-w):
compounds were also tested against Gram-positive bac- (1.36 mmol) in CH3CN (10 ml), K2CO3 (6.79 mmol) was
teria Bacillus anthracis (Sterne strain). With the limited added and the resulting mixture was stirred for 10-15 min.
data set, it seems that the potency against B. anthracis To this was added the appropriate (bromomethyl)phenyl
was comparable with or higher than that of E. coli. For derivatives (1.22 mmol) and the reaction was stirred at
example, the MIC of 3w was 25 µM against E. coli and room temperature for 16-18h. Upon disappearance of the
12.5 µM against B. anthracis. By examining the details, starting material, the solvent was removed in vacuo. The
it seems that a CF3 group at the R2 position is beneficial, dried residue was washed by H2O (pH = 9-10, 20 mL × 2)
especially if the central linker is –CH2-O-. With a -O- linker, and brine (15 ml × 2) followed by product extraction in
it seems that an ester group also offers a good outcome. ethyl acetate (20 ml). The solvent was evaporated in vacuo
Overall, the results are very useful information for those to obtain crude product, which was purified using silica
who might be interested in SecA inhibitor for both impro- gel column chromatography.
ved potencies and for avoiding chemical space that would
not be productive.
2-mercapto-4-(4-((4-methylbenzyl)oxy)phenyl)-6-oxo-
1,6-dihydropyrimidine-5-carbonitrile (2b)
Conclusion
Yield 37%; white solid; 1H NMR (DMSO-d6): δ 13.16 (bs, 1H),
12.86 (s, 1 H), 7.66 (d, J = 8.8 Hz, 2H), 7.37 (d, J = 8.1 Hz, 2H),
We have described the design, syntheses and biological
7.22 (d, J = 7.6 Hz, 2H), 7.16 (d, J = 8.4 Hz, 2H), 5.16 (s, 2H); 13C
evaluation of a novel structural class of small-molecule
NMR (DMSO-d6): δ 177.5, 161.7, 159.7, 137.8, 133.9, 131.2, 129.5,
SecA inhibitors with µM inhibition. A CF3 group at the R2
128.4, 122.8, 116.1, 115.1, 89.5, 69.9, 21.3. HRMS (ESI) (m/z):
position and a diaryl ether linker seem to be beneficial for
Calcd. for C19H14N3O2S, [M-H]+: 348.0803; found: 348.0803.
improved potency of the lead compound.
2-mercapto-4-(4-((4-methoxybenzyl)oxy)phenyl)-6-oxo-
1,6-dihydropyrimidine-5-carbonitrile (2c)
Experimental
All chemical reagents and solvents used were reagent Yield 42%; white solid; 1H NMR (DMSO-d6): δ 13.15 (bs, 1H),
grade or purified using standard methods. TLC analy- 12.85 (s, 1H), 7.66 (d, J = 8.8 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H),
ses were conducted on silica gel plates (Sorbent Silica G 7.16 (d, J = 8.4 Hz, 2H), 6.96 (d, J = 8.4 Hz, 2H), 5.12 (s, 2H),

F. Bamba et al.: Design of novel SecA inhibitorsꢁ
ꢁ79
3.76 (s, 3H); 13C NMR (DMSO-d6): δ 177.4, 161.7, 161.6, 159.6, 4-(5-cyano-2-mercapto-6-oxo-1,6-dihydropyrimidin-
4-yl)-N-(p-tolyl)benzamide (2i)
159.6, 131.2, 130.1, 128.7, 22.7, 116.1, 115.0, 114.3, 89.4, 69.7,
55.5. HRMS (ESI) (m/z): Calcd. for C19H14N3O3S, [M-H]+:
364.0750; found: 364.0753.
Yield 34%; white solid; 1H NMR (DMSO-d6): δ 11.68 (s, 1H),
10.29 (s, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.86 (d, J = 8.4 Hz,
2H), 7.68 (d, J = 8.4 Hz, 2H), 7.17 (d, J = 8.4Hz, 2H); 13C NMR
(DMSO-d6): δ 183.6, 167.0, 165.2, 162.7, 140.8, 137.0, 136.6,
133.2, 129.4, 128.6, 127.7, 120.9, 119.1, 86.0, 20.9. HRMS (ESI)
(m/z): Calcd. for C19H13N4O2S, [M-H]+: 361.0754; found:
4-(4-((4-fluorobenzyl)oxy)phenyl)-2-mercapto-6-oxo-
1,6-dihydropyrimidine-5-carbonitrile (2d)
Yield 40%; white solid; 1H NMR (DMSO-d6): δ 13.17 (bs, 1H), 361.0763.
12.84 (s, 1H), 7.68 (d, J = 8.0 Hz, 2H), 7.54 (m, 2H), 7.24 (t, J =
13
8.8 Hz, 2H), 7.18 (d, J = 8.8 Hz, 2H), 5.19 (s, 2H); C NMR
(DMSO-d6): δ 177.4, 163.6, 161.4, 159.5, 133.2, 131.1, 130.6, 4-(4-(benzyloxy)phenyl)-6-oxo-2-((4-(trifluoromethyl)
benzyl)thio)-1,6-dihydropyrimidine-5-carbonitrile (3a)
115.8, 115.6, 114.9, 89.1, 69.2. HRMS (ESI) (m/z): Calcd. for
C18H12FN3O2S, [M-H]+: 352.0551; found: 352.0556.
Yield 54%; white solid; 1H NMR (DMSO-d6): δ 7.93 (d, J = 8.4
Hz, 2H), 7.66 (qd, J = 8.0 Hz, 4H), 7.48 (d, J = 7.2 Hz, 2H), 7.41
(t, J = 7.2 Hz, 1H), 7.37(m, 1H), 7.18 (d, J = 8.4 Hz, 2H), 5.22 (s,
2-mercapto-6-oxo-4-(4-((4-(trifluoromethyl)benzyl)oxy)
phenyl)-1,6-dihydropyrimidine-5-carbonitrile (2e)
13
2H), 4.61 (s, 2H); C NMR (DMSO-d6): δ 166.7, 165.5, 161.6,
142.5, 136.9, 131.2, 130.1, 128.9, 128.4, 128.3, 127.9, 126.0,
1
Yield 36%; white solid; H NMR (DMSO-d6): δ 13.18 (bs, 125.8, 125.7, 123.3, 116.9, 115.2, 91.9, 69.9, 33.8. HRMS (ESI)
1H), 12.73 (s, 1H), 7.79 (d, J = 8.0 Hz, 4H), 7.70 (m, 4H), (m/z): Calcd. for C26H17F3N3O2S, [M-H]+: 492.0994; found:
7.19 (d, J = 8.8 Hz, 2H), 5.34 (s, 2H); 13C NMR (DMSO-d6): δ 492.0998.
177.8, 161.9, 161.2, 159.9, 141.9, 131.2, 129,1, 128.7, 128.6, 126.0,
125.8, 125.8, 123.6, 123.3, 116.3, 115.0, 89.2, 69.0. HRMS (ESI)
(m/z): Calcd. for C19H11F3N3O2S, [M-H]+: 402.0519; found: 2-((4-azidobenzyl)thio)-4-(4-((4-methylbenzyl)oxy)
phenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile
(3b)
402.0522.
4-(5-cyano-2-mercapto-6-oxo-1,6-dihydropyrimidin-
4-yl)-N-phenylbenzamide (2g)
Yield 61%; white solid; 1H NMR (DMSO-d6): δ 8.00 (d, J =
8.4 Hz, 2H), 7.46 (d, J = 8.0 Hz, 2H), 7.37 (d, J = 8.0 Hz,
2H), 7.20 (t, J = 8.8 Hz, 4H), 7.07 (d, J = 8.4 Hz, 2H), 5.17 (s,
Yield 42%; white solid; 1H NMR (DMSO-d6): δ 12.32 (s, 1H), 2H), 4.54 (s, 2H), 2.31 (s, 3H); 13C NMR (DMSO-d6): δ 166.8,
10.41 (s, 1H), 8.07 (d, J = 8.4 Hz, 2H), 7.85 (d, J = 8.0 Hz, 2H), 165.4, 161.8, 139.0, 137.7, 133.9, 133.9, 131.2, 131.1, 129.5,
7.80 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 7.10 (t, J = 128.3, 127.7, 119.6, 116.7, 115.3, 92.0, 69.9, 34.0, 21.2. HRMS
7.2 Hz, 2H); 13C NMR (DMSO-d6): δ 180.1, 167.6, 165.3, 163.9, (ESI) (m/z): Calcd. for C26H19N6O2S, [M-H]+: 479.1290;
160.9, 141.1, 139.4, 137.5, 136.6, 129.5, 129.1, 129.0, 128.2, found: 479.1300.
128.0, 124.3, 120.9, 120.8, 118.6, 117.0, 88.8. HRMS (ESI)
(m/z): Calcd. for C18H11N4O2S, [M-H]+: 347.0597; found:
347.0599.
methyl4-(((5-cyano-4-(4-((4-methylbenzyl)oxy)phenyl)-
6-oxo-1,6-dihydropyrimidin-2-yl)thio)methyl)benzoate
(3c)
N-(4-chlorophenyl)-4-(5-cyano-2-mercapto-6-oxo-1,6-di-
hydropyrimidin-4-yl)benzamide (2h)
1
Yield 55%; white solid; H NMR (DMSO-d6): δ 7.94 (d, J =
8.0 Hz, 2H), 7.90 (d, J = 8.0 Hz, 2H), 7.57 (d, J = 7.6 Hz, 2H),
Yield 38%; white solid; 1H NMR (DMSO-d6): δ 11.76 (s, 1H), 7.36 (d, J = 7.6 Hz, 2H), 7.21 (d, J = 7.6 Hz, 2H), 7.17 (d, J = 8.4
10.52 (s, 1H), 8.03 (d, J = 8.0 Hz, 2H), 7.86 (m, 4H), 7.42 (d, Hz, 2H), 5.17 (s, 2H), 4.60 (s, 2H), 3.83 (s, 3H), 2.31 (s, 3H);
13
J = 8.8 Hz, 2H); C NMR (DMSO-d6): δ 183.1, 166.6, 165.5, 13C NMR (DMSO-d6): δ 166.8, 166.4, 165.5, 161.7, 137.7, 133.9,
162.7, 140.3, 138.5, 136.3, 128.9, 128.7, 127.9, 122.4, 118.8, 131.2, 129.8, 129.7, 129.5, 129.0, 128.4, 127.8, 115.2, 91.8, 69.9,
86.2. HRMS (ESI) (m/z): Calcd. for C18H10ClN4O2S, [M-H]+: 52.5, 34.1, 21.2. HRMS (ESI) (m/z): Calcd. for C28H22N3O4S,
381.0208; found: 381.0209.
[M-H]+: 496.1331; found: 496.1337.

ꢁ
80ꢁ
F. Bamba et al.: Design of novel SecA inhibitors
2-((4-azidobenzyl)thio)-4-(4-((4-methoxybenzyl)oxy)
phenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (3d)
Methyl 4-(((5-cyano-4-(4-((4-fluorobenzyl)oxy)phenyl)-
6-oxo-1,6-dihydropyrimidin-2-yl)thio)methyl)benzoate
(3h)
1
Yield 50%; white solid; H NMR (DMSO-d6): δ 8.00 (d, J =
1
8.4 Hz, 2H), 7.46 (d, J = 8.0 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), Yield 63%; white solid; H NMR (DMSO-d6): δ 7.94 (d, J =
7.19 (d, J = 8.4 Hz, 2H), 7.07 (d, J = 8.4 Hz, 2H), 6.96 (d, J = 8.8 Hz, 2H), 7.89 (d, J = 8.0 Hz, 4H), 7.52 - 7.58 (m, 4H), 7.25
8.4 Hz, 2H), 5.14 (s, 2H), 4.54 (s, 2H), 3.76 (s, 3H); 13C NMR (t, J = 8.8 Hz, 2H), 7.17 (d, J = 8.8 Hz, 2H), 5.20 (s, 2H), 4.58
(DMSO-d6): δ 168.1, 166.6, 165.3, 161.9, 161.7, 159.6, 139.0, (s, 2H), 3.84 (s, 3H); 13C NMR (DMSO-d6): δ 166.6, 166.4,
133.9, 131.2, 131.1, 130.1, 128.8, 127.7, 119.7, 116.7, 115.3, 115.0, 163.5, 161.4, 143.4, 133.2, 131.1, 130.6, 130.5, 129.7, 129.7, 129.0,
114.3, 92.0, 69.8, 55.5, 34.0. HRMS (ESI) (m/z): Calcd. for 128.2, 117.3, 115.8, 115.6, 115.1, 90.6, 69.2, 52.5, 34.1. HRMS
C26H19N6O3S, [M-H]+: 495.1239; found: 495.1249.
(ESI) (m/z): Calcd. for C27H19FN3O4S, [M-H]+: 500.1080;
found:500.1093.
Methyl4-(((5-cyano-4-(4-((4-methoxybenzyl)oxy)phenyl)-
6-oxo-1,6-dihydropyrimidin-2-yl)thio)methyl)benzoate (3e)
4-(4-((4-fluorobenzyl)oxy)phenyl)-6-oxo-2-((4-
(trifluoromethyl)benzyl)thio)-1,6-dihydropyrimidine-
5-carbonitrile (3i)
1
Yield 49%; white solid; H NMR (DMSO-d6): δ 7.95 (d, J =
8.8 Hz, 2H), 7.90 (d, J = 8.0 Hz, 4H), 7.57 (d, J = 8.0 Hz, 2H),
1
7.41 (d, J = 8.4 Hz, 2H), 7.17 (d, J = 8.4 Hz, 2H), 6.96 (d, J = Yield 66%; white solid; H NMR (DMSO-d6): δ 7.92 (d, J =
8.4 Hz, 2H), 5.13 (s, 2H), 4.60 (s, 2H), 3.83 (s, 3H), 3.76 (s, 8.0 Hz, 2H), 7.94 (qd, J = 8.0 Hz, 4H), 7.53 (t, J = 6.4 Hz, 2H),
3H); 13C NMR (DMSO-d6): δ 166.6, 166.3, 165.2, 161.8, 159.6, 7.23 (t, J = 8.4 Hz, 2H), 7.17 (d, J = 8.4 Hz, 2H), 5.19 (s, 2H),
142.9, 131.2, 130.1, 128.8, 129.7, 129.1, 128.8, 127.6, 115.1, 114.3, 4.58 (s, 2H); 13C NMR (DMSO-d6): δ 166.7, 166.2, 163.5, 161.4,
92.0, 69.8, 55.5, 52.6, 34.1. HRMS (ESI) (m/z): Calcd. for 161.1, 142.8, 133.2, 132.5, 131.1, 130.6, 130.5, 130.1, 128.2,
C28H22N3O5S, [M-H]+: 512.1280; found: 522.1299.
125.7, 117.3, 115.8, 115.6, 115.1, 90.6, 69.2, 33.8. HRMS (ESI)
(m/z): Calcd. for C26H16F4N3O2S, [M-H]+: 510.0899; found:
510.0896.
4-(4-((4-methoxybenzyl)oxy)phenyl)-6-oxo-2-((4-
(trifluoromethyl)benzyl)thio)-1,6-dihydropyrimidine-
5-carbonitrile (3f)
2-((4-azidobenzyl)thio)-6-oxo-4-(4-((4-(trifluoromethyl)
benzyl)oxy)phenyl)-1,6-dihydropyrimidine-5-carbonitrile
(3j)
1
Yield 54%; white solid; H NMR (DMSO-d6): δ 7.92 (d, J =
8.4 Hz, 2H), 7.66 (qd, J = 8.0 Hz, 4H), 7.41 (d, J = 8.4 Hz, 2H),
1
7.16 (d, J = 8.8 Hz, 2H), 6.96 (d, J = 8.8 Hz, 2H), 5.12 (s, 2H), Yield 71%; white solid; H NMR (DMSO-d6): δ 8.01 (d, J =
4.60 (s, 2H), 3.73 (s, 3H); 13C NMR (DMSO-d6): δ 166.6, 161.7, 8.8 Hz, 2H), 7.79 (d, J = 8.0 Hz, 2H), 7.70 (d, J = 8.0 Hz, 2H),
159.6, 142.6, 131.3, 130.1, 128.8, 128.5, 128.2, 127.9, 126.0, 7.45 (d, J = 8.0 Hz, 2H), 7.22 (d, J = 8.4 Hz, 2H), 7.06 (d, J =
13
125.8, 125.7, 117.1, 115.2, 114.3, 91.7, 69.7, 55.5, 30.9. HRMS 8.4 Hz, 2H), 5.36 (s, 2H), 4.53 (s, 2H); C NMR (DMSO-d6):
(ESI) (m/z): Calcd. for C27H19F3N3O3, [M-H]+: 522.1099; δ 161.4, 141.8, 139.0, 133.9, 131.3, 131.1, 129.1, 128.6, 128.1,
found: 522.1090.
125.8, 119.6, 116.7, 115.3, 115.1, 92.1, 69.0, 34.0. HRMS (ESI)
(m/z): Calcd. for C26H16F3N6O2S, [M-H]+: 533.1008; found:
533.1001.
2-((4-azidobenzyl)thio)-4-(4-((4-fluorobenzyl)oxy)
phenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (3g)
Methyl 4-(((5-cyano-6-oxo-4-(4-((4-(trifluoromethyl)
benzyl)oxy)phenyl)-1,6-dihydropyrimidin-2-yl)thio)
methyl)benzoate (3k)
1
Yield 47%; white solid; H NMR (DMSO-d6): δ 8.00 (d, J =
8.0 Hz, 2H), 7.54 (t, J = 7.2 Hz, 4H), 7.46 (d, J = 7.6 Hz, 2H),
7.25 (d, J = 8.4 Hz, 2H), 7.20 (d, J = 8.8 Hz, 2H), 7.07 (d, J =
13
1
7.6 Hz, 2H), 5.21 (s, 2H), 4.54 (s, 2H); C NMR (DMSO-d6): Yield 52%; white solid; H NMR (DMSO-d6): δ 7.96 (d, J =
δ 166.6, 165.5, 163.5, 161.8, 161.6, 161.1, 139.1, 133.9, 133.2, 8.0 Hz, 2H), 7.89 (d, J = 8.0 Hz, 2H), 7.79 (d, J = 7.6 Hz,
133.1, 131.3, 131.1, 130.6, 130.5, 127.9, 119.6, 116.7, 115.9, 115.6, 2H), 7.70 (d, J = 7.6 Hz, 2H), 7.56 (d, J = 8.0 Hz, 2H), 7.20
115.3, 115.1, 92.0, 69.3, 34.0. HRMS (ESI) (m/z): Calcd. for (d, J = 8.0 Hz, 2H), 5.76 (s, 2H), 4.60 (s, 2H), 3.83 (s, 3H);
C25H16FN6O2S, [M-H]+: 483.1039; found: 483.1031.
13C NMR (DMSO-d6): δ 166.4, 161.4, 142.9, 141.9, 131.3, 129.7,

F. Bamba et al.: Design of novel SecA inhibitorsꢁ
ꢁ81
129.7, 129.1, 128.6, 125.8, 125.8, 115.3, 92.1, 69.0, 55.3, 52.5, 7.37 (t, J = 8.0 Hz, 2H), 7.12 (t, J = 7.6 Hz, 1H), 4.57 (s, 2H),
34.1. HRMS (ESI) (m/z): Calcd. for C28H19F3N3O4S, [M-H]+: 3.83 (s, 3 H); 13C NMR (DMSO-d6): δ 167.7, 167.0, 166.4, 163.6,
550.1048; found: 550.1039.
143.4, 139.4, 138.8, 137.7, 129.7, 129.1, 129.0, 128.9, 128.2, 124.4,
120.8, 117.0, 93.4, 52.5, 34.1. HRMS (ESI) (m/z): Calcd. for
C27H19N4O4S, [M-H]+: 495.1127; found: 495.1132.
6-oxo-4-(4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-2-
((4-(trifluoromethyl)benzyl)thio)-1,6-dihydropyrimidine-
5-carbonitrile (3l)
4-(5-cyano-6-oxo-2-((4-(trifluoromethyl)benzyl)thio)-
1,6-dihydropyrimidin-4-yl)-N-phenylbenzamide (3p)
1
Yield 68%; white solid; H NMR (DMSO-d6): δ7.94 (d, J =
8.0 Hz, 2H), 7.78 (d, J = 8.0 Hz, 2H), 7.70 (d, J = 8.0 Hz, 2H), Yield 48%; white solid; 1H NMR (DMSO-d6): δ 10.41 (s, 1H),
7.66 (qd, J = 8.4 Hz, 4H), 7.20 (d, J = 8.8 Hz, 2H), 5.35 (s, 8.08 (d, J = 8.0 Hz, 2H), 7.97 (d, J = 8.4 Hz, 2H), 7.79 (d, J =
2H), 4.61 (s, 2H); 13C NMR (DMSO-d6): δ 166.7, 165.3, 161.4, 8.0 Hz, 2H), 7.67 (qd, J = 8.0 Hz, 4H), 7.37 (t, J = 8.0 Hz, 2H),
13
142.4, 141.8, 131.3, 130.1, 129.1, 128.7, 128.2, 128.0, 125.8, 7.12 (t, J = 7.6 Hz, 1H), 4.56 (s, 2H); C NMR (DMSO-d6): δ
125.8, 125.7, 123.3, 116.7, 115.2, 92.2, 69.0, 33.9. HRMS (ESI) 167.7, 167.0, 165.3, 164.1, 143.0, 139.4, 138.9, 137.6, 130.1, 129.1,
(m/z): Calcd. for C27H17F6N3O2S, [M-H]+: 560.0867; found: 129.0, 128.2, 125.7, 124.3, 123.3, 120.8, 117.1, 93.4, 33.9. HRMS
560.0870.
(ESI) (m/z): Calcd. for C26H16F3N4O2S, [M-H]+: 505.0946;
found: 505.0941.
6-oxo-4-(4-phenoxyphenyl)-2-((4-(trifluoromethyl)
benzyl)thio)-1,6-dihydropyrimidine-5-carbonitrile (3m)
4-(2-((4-azidobenzyl)thio)-5-cyano-6-oxo-1,6-dihydropy-
rimidin-4-yl)-N-(4-chlorophenyl)benzamide (3q)
1
Yield 84%; white solid; H NMR (DMSO-d6): δ 7.93 (d, J =
8.0 Hz, 2H), 7.66 (qd, J = 8.0 Hz, 4H), 7.47 (t, J = 7.2 Hz, 2H), Yield 18%; white solid; 1H NMR (DMSO-d6): δ 10.56 (s, 1H),
7.24 (t, J = 7.2 Hz, 1H), 7.14 (d, J = 7.6 Hz, 2H), 7.10 (d, J = 7.6 8.07 (d, J = 8.4 Hz, 2H), 7.98 (d, J = 8.0 Hz, 2H), 7.85 (d, J =
13
Hz, 2H), 4.58 (s, 2H); C NMR (DMSO-d6): δ 166.6, 166.3, 8.8 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.8 Hz, 2H),
13
162.8, 160.4, 155.6, 142.7, 131.4, 130.7, 130.2, 130.1, 128.4, 7.07 (d, J = 8.4 Hz, 2H), 4.44 (s, 2H); C NMR (DMSO-d6):
128.1, 126.0, 125.8, 125.7, 125.1, 123.3, 120.3, 117.7, 117.0, 90.3, δ 168.5, 166.9, 165.4, 164.7, 139.3, 138.7, 138.5, 137.1, 134.9,
33.8. HRMS (ESI) (m/z): Calcd. for C25H15F3N3O2S, [M-H]+: 133.6, 131.0, 129.0, 128.2, 127.8, 127.1, 125.5, 123.9, 122.3,
478.0837; found: 478.0828.
119.7, 119.5, 117.5, 113.8, 111.7, 92.7, 33.9. HRMS (ESI) (m/z):
Calcd. for C25H15ClN7O2S, [M-H]+: 512.0696; found: 512.0698.
4-(2-((4-azidobenzyl)thio)-5-cyano-6-oxo-1,6-dihydropy-
rimidin-4-yl)-N-phenylbenzamide (3n)
Methyl4-(((4-(4-((4-chlorophenyl)carbamoyl)phenyl)-
5-cyano-6-oxo-1,6-dihydropyrimidin-2-yl)thio)methyl)
benzoate (3r)
Yield 65%; white solid; 1H NMR (DMSO-d6): δ 10.41 (s, 1H),
8.08 (d, J = 8.4 Hz, 2H), 8.01 (d, J = 8.0 Hz, 2H), 7.79 (d, J =
8.0 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 7.37 (t, J = 8.0 Hz, 2H),
7.12 (t, J = 7.6 Hz, 1H), 7.07 (d, J = 8.0 Hz, 2H), 4.48 (s, 2H);
13C NMR (DMSO-d6): δ 167.8, 167.1, 165.5, 163.0, 139.4, 138.8,
137.8, 134.6, 131.1, 129.1, 128.2, 124.6, 120.8, 119.6, 117.0, 93.4,
43.0. HRMS (ESI) (m/z): Calcd. for C25H16N7O2S, [M-H]+:
478.1086; found: 478.1099.
Yield 31%; white solid; 1H NMR (DMSO-d6): δ 10.55 (s, 1H),
8.08 (d, J = 8.4 Hz, 2H), 7.98 (d, J = 8.4 Hz, 2H), 7.90 (d, J =
8.0 Hz, 2H), 7.84 (d, J = 8.8 Hz, 2H), 7.57 (d, J = 8.0 Hz, 2H),
13
7.43 (d, J = 8.8 Hz, 2H), 4.57 (s, 2H), 3.84 (s, 3H); C NMR
(DMSO-d6): δ 167.6, 166.9, 166.4, 165.4, 163.7, 143.5, 139.0,
138.4, 137.3, 129.7, 129.3, 129.2, 129.1, 129.0, 128.9, 128.2, 127.9,
122.3, 117.0, 93.3, 52.58, 34.1. HRMS (ESI) (m/z): Calcd. for
C27H18ClN4O4S, [M-H]+: 529.0737; found: 529.0734.
Methyl4-(((5-cyano-6-oxo-4-(4-(phenylcarbamoyl)
phenyl)-1,6-dihydropyrimidin-2-yl)thio)methyl)benzoate
(3o)
N-(4-chlorophenyl)-4-(5-cyano-6-oxo-2-((4-
(trifluoromethyl)benzyl)thio)-1,6-dihydropyrimidin-4-yl)
benzamide (3s)
Yield 54%; white solid; 1H NMR (DMSO-d6): δ 10.41 (s, 1H),
8.08 (d, J = 8.4 Hz, 2H), 7.98 (d, J = 8.4 Hz, 2H), 7.91 (d, J = Yield 17%; white solid; H NMR (DMSO-d6): δ 10.55 (s,
8.0 Hz, 2H), 7.79 (d, J = 8.0 Hz, 2H), 7.57 (d, J = 8.0 Hz, 2H), 1H), 8.05 (d, J = 8.0 Hz, 2H), 7.94 (d, J = 8.4 Hz, 2H), 7.84
1

ꢁ
82ꢁ
F. Bamba et al.: Design of novel SecA inhibitors
(d, J = 8.8 Hz, 2H), 7.67 (qd, J = 8.0 Hz, 4H), 7.57 (d, J = was conducted with the partial financial support of the
13
8.0 Hz, 2H), 7.43 (d, J = 8.8Hz, 2H), 4.52 (s, 2H); C NMR National Institute of Health awarded to PCT and BW
(DMSO-d6): δ 168.2, 166.9, 165.4, 143.1, 139.1, 138.4, 137.30, (AI104168).
130.1, 129.0,128.2, 127.9, 127.1, 126.0, 125.8, 125.7, 122.3, 117.2,
93.1, 33.8. HRMS (ESI) (m/z): Calcd. for C26H15ClF3N4O2S,
[M-H]+: 539.0556; found: 539.0541.
References
[1] Ventola CL. The antibiotic resistance crisis: part 1: causes and
4-(2-((4-azidobenzyl)thio)-5-cyano-6-oxo-1,6-dihydropy-
threats. P&T. 2015; 40: 277–83.
rimidin-4-yl)-N-(p-tolyl)benzamide (3t)
[2] Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME,
Giske CG, et al. Multidrug-resistant, extensively drug-resistant
Yield 51%; white solid; 1H NMR (DMSO-d6): δ 10.34 (s, 1H),
and pandrug-resistant bacteria: an international expert
proposal for interim standard definitions for acquired
resistance. Clin Microbiol Infect. 2012; 18: 268–81.
[3] Willyard C. The drug-resistant bacteria that pose the greatest
health threats. Nature. 2017; 543:15.
[4] Antibiotic / Antimicrobial Resistance (AR / AMR) https://
http://www.cdc.gov/drugresistance/index.html
[5] Zimmer J, Nam Y, Rapoport TA. Structure of a complex of the
ATPase SecA and the protein-translocation channel. Nature.
2008; 455: 936–43.
8.08 (d, J = 8.4 Hz, 2H), 8.03 (d, J = 8.4 Hz, 2H), 7.67 (d, J
= 8.0 Hz, 2H), 7.46 (d, J = 8.0 Hz, 2H), 7.17 (d, J = 8.4 Hz,
13
2H), 7.07 (d, J = 8.0 Hz, 2H), 4.51 (s, 2H); C NMR (DMSO-
d6): δ 167.1, 165.2, 138.9, 137.8, 136.7, 134.6, 133.5, 129.5, 129.0,
128.2, 120.8, 120.0, 119.6, 116.7, 93.4, 34.0, 20.9. HRMS (ESI)
(m/z): Calcd. for C26H18N7O2S, [M-H]+: 492.1243; found:
492.1250.
[6] Driessen AJ, Fekkes P, van der Wolk JP. The Sec system. Curr
Opin Microbiol. 1998; 1: 216–22.
[7] Hsieh YH, Zhang H, Lin BR, Cui N, Na B, Yang H, et al. SecA
alone can promote protein translocation and ion channel
activity: SecYEG increases efficiency and signal peptide
specificity. J Biol Chem. 2011; 286: 44702–9.
Methyl-4-(((5-cyano-6-oxo-4-(4-(p-tolylcarbamoyl)
phenyl)-1,6-dihydropyrimidin-2-yl)thio)methyl)benzoate
(3u)
[8] Wang H, Ma Y, Hsieh YH, Yang H, Li M, Wang B, et al. Trimer Is
Functional in the Membranes: Existence of Higher Oligomers?
Biochem. Biophys. Res Commun. 2014; 447: 250–4.
[9] Chaudhary AS, Chen W, Jin J, Tai PC, Wang B. SecA: a potential
antimicrobial target. Future Med Chem. 2015; 7: 989–1007.
[10] Sugie Y, Inagaki S, Kato Y, Nishida H, Pang CH, Saito T, et al.
CJ-21,058, a new SecA inhibitor isolated from a fungus.
J Antibiot (Tokyo). 2002; 55: 25–9.
[11] Parish CA, de la Cruz M, Smith SK, Zink D, Baxter J, Tucker-
Samaras S, et al. Antisense-guided isolation and structure
elucidation of pannomycin, a substituted cis-decalin from
Geomyces pannorum. J Nat Prod. 2009; 72: 59–62.
[12] Jang MY, De Jonghe S, Segers K, Anne J, Herdewijn P. Synthesis
of novel 5-amino-thiazolo[4,5-d]pyrimidines as E. coli and S.
aureus SecA inhibitors. Bioorg Med Chem. 2011; 19: 702–14.
[13] Akula N, Trivedi P, Han FQ, Wang N. Identification of small
molecule inhibitors against SecA of Candidatus Liberibacter
asiaticus by structure based design. Eur J Med Chem. 2012;
54: 919–24.
1
Yield 62%; white solid; H NMR (DMSO-d6): δ 10.34 (s,
1H), 8.07 (d, J = 8.0 Hz, 2H), 7.98 (d, J = 8.0 Hz, 2H), 7.91
(d, J = 8.0 Hz, 2H), 7.67 (d, J = 8.0 Hz, 2H), 7.58 (d, J =
8.4 Hz, 2H), 7.17 (d, J = 8.0 Hz, 2H), 4.58 (s, 2H), 3.83 (s,
3H); 13C NMR (DMSO-d6): δ 167.5, 167.0, 166.4, 165.1, 143.5,
138.7, 137.7, 136.9, 133.3, 132.1, 129.9, 129.7, 129.5, 129.0,
128.9, 128.1, 117.0, 93.3, 52.5, 34.1, 20.9. HRMS (ESI)
(m/z): Calcd. for C28H21N4O4S, [M-H]+: 509.1278; found:
509.1277.
4-(5-cyano-6-oxo-2-((4-(trifluoromethyl)benzyl)thio)-
1,6-dihydropyrimidin-4-yl)-N-(p-tolyl)benzamide (3v)
Yield 45%; white solid; 1H NMR (DMSO-d6): δ 10.34 (s, 1H),
8.07 (d, J = 8.4 Hz, 2H), 7.97 (d, J = 8.4 Hz, 2H), 7.64 – 7.71 (m,
6H), 7.16 (d, J = 8.4 Hz, 2H), 4.59 (s, 2H); C NMR (DMSO-
d6): δ 167.1, 167.0, 165.1, 162.8, 142.7, 138.5, 137.8, 136.9, 133.3,
130.2, 129.4, 129,0, 128.5, 128.2, 125.7, 123.5, 120.8, 116.7, 93.7,
33.9, 20.9. HRMS (ESI) (m/z): Calcd. for C27H18F3N4O2S,
[M-H]+: 519.1108; found: 519.1094.
[14] Akula N, Zheng H, Han FQ, Wang N. Discovery of novel SecA
inhibitors of Candidatus Liberibacter asiaticus by structure
based design. Bioorg Med Chem Lett. 2011; 21:
13
4183–8.
[15] Li M, Huang YJ, Tai PC, Wang B. Discovery of the first SecA
inhibitors using structure-based virtual screening. Biochem
Biophys Res Commun. 2008; 368: 839–45.
[16] Chen W, Huang YJ, Gundala SR, Yang H, Li M, Tai PC, et al.
The first low μM SecA inhibitors. Bioorg Med Chem. 2010;
18: 1617–25.
[17] Huang YJ, Wang H, Gao FB, Li M, Yang H, Wang B, et al.
Fluorescein analogues inhibit SecA ATPase: the first
Acknowledgments: FB was a visiting scholar at GSU
when conducting the lab research with partial financial
support from the Islamic Development Bank (IDB) under
a merit scholarship programme. Part of the research

F. Bamba et al.: Design of novel SecA inhibitorsꢁ
ꢁ83
sub-micromolar inhibitor of bacterial protein translocation.
ChemMedChem. 2012; 7: 571–7.
effect in alpha-MSH-stimulated B16F10 melanoma cells. Eur
J Med Chem. 2019; 161: 78–92.
[21] Arienti KL, Brunmark A, Axe FU, McClure K, Lee A, Blevitt J,
et al. Checkpoint kinase inhibitors: SAR and radioprotective
properties of a series of 2-arylbenzimidazoles. J Med Chem.
2005; 48: 1873–85.
[22] Cui J, Jin J, Chaudhary AS, Hsieh YH, Zhang H, Dai C, et al.
Design, Synthesis and Evaluation of Triazole-Pyrimidine
Analogues as SecA Inhibitors. ChemMedChem. 2016; 11:
43–56.
[18] Cui J, Jin J, Hsieh YH, Yang H, Ke B, Damera K, et al. Design,
synthesis and biological evaluation of rose bengal analogues
as SecA inhibitors. ChemMedChem. 2013; 8: 1384–93.
[19] Chaudhary AS, Jin J, Chen W, Tai PC, Wang B. Design, syntheses
and evaluation of 4-oxo-5-cyano thiouracils as SecA inhibitors.
Bioorg Med Chem. 2015; 23: 105–17.
[20] Ullah S, Kang D, Lee S, Ikram M, Park C, Park Y, et al. Synthesis
of cinnamic amide derivatives and their anti-melanogenic