Design, Synthesis and Evaluation of Triazole-Pyrimidine Analogues as SecA Inhibitors

Article abstract of DOI:10.1002/cmdc.201500447

SecA, a key component of the bacterial Sec-dependent secretion pathway, is an attractive target for the development of new antimicrobial agents. Through a combination of virtual screening and experimental exploration of the surrounding chemical space, we identified a hit bistriazole SecA inhibitor, SCA-21, and studied a series of analogues by systematic dissections of the core scaffold. Evaluation of these analogues allowed us to establish an initial structure-activity relationship in SecA inhibition. The best compounds in this group are potent inhibitors of SecA-dependent protein-conducting channel activity and protein translocation activity at low- to sub-micromolar concentrations. They also have minimal inhibitory concentration (MIC) values against various strains of bacteria that correlate well with the SecA and protein translocation inhibition data. These compounds are effective against methicillin-resistant Staphylococcus aureus strains with various levels of efflux pump activity, indicating the capacity of SecA inhibitors to null the effect of multidrug resistance. Results from studies of drug-affinity-responsive target stability and protein pull-down assays are consistent with SecA as a target for these compounds. One stone, several birds! The SecA inhibitors discovered in this study are broad-spectrum antimicrobials with the intrinsic ability to null the effect of efflux pumps. They are therefore effective against multidrug-resistant bacterial strains, can inhibit virulence factor secretion, and are very active against strains of bacteria that are resistant to antibiotics in current use, including vancomycin.

Full text of DOI:10.1002/cmdc.201500447

DOI: 10.1002/cmdc.201500447
Full Papers
Design, Synthesis and Evaluation of Triazole-Pyrimidine
Analogues as SecA Inhibitors
Jianmei Cui,[a] Jinshan Jin,[b] Arpana Sagwal Chaudhary,[a] Ying-hsin Hsieh,[b] Hao Zhang,[b]
Chaofeng Dai,[a] Krishna Damera,[a] Weixuan Chen,[a] Phang C. Tai,*[b] and Binghe Wang*[a]
SecA, a key component of the bacterial Sec-dependent secre-
tion pathway, is an attractive target for the development of
new antimicrobial agents. Through a combination of virtual
screening and experimental exploration of the surrounding
chemical space, we identified a hit bistriazole SecA inhibitor,
SCA-21, and studied a series of analogues by systematic dissec-
tions of the core scaffold. Evaluation of these analogues al-
lowed us to establish an initial structure–activity relationship in
SecA inhibition. The best compounds in this group are potent
inhibitors of SecA-dependent protein-conducting channel ac-
tivity and protein translocation activity at low- to sub-micro-
molar concentrations. They also have minimal inhibitory con-
centration (MIC) values against various strains of bacteria that
correlate well with the SecA and protein translocation inhibi-
tion data. These compounds are effective against methicillin-
resistant Staphylococcus aureus strains with various levels of
efflux pump activity, indicating the capacity of SecA inhibitors
to null the effect of multidrug resistance. Results from studies
of drug-affinity-responsive target stability and protein pull-
down assays are consistent with SecA as a target for these
compounds.
Introduction
Because of drug resistance issues, bacterial pathogens have re-
emerged as a serious public health concern.[1] There is an
urgent need to develop new antimicrobials, especially those
with novel mechanisms of action. Common antibiotic mecha-
nisms include inhibition of essential processes in the bacterium
to disrupt replication, transcription, translation, or cell-wall syn-
thesis.[2] Recently, SecA, a key enzyme in protein transloca-
tion,[3] has drawn interest in the development of antimicrobial
agents[4] because of its unique role in bacterial survival, viru-
lence factor secretion, and membrane integration of certain
efflux pumps, which are responsible for multidrug resistance.[5]
In both Gram-positive and Gram-negative bacteria, the ma-
jority of secretory proteins, including virulence factors, are
translocated across the cytoplasmic membrane through the
Sec-dependent pathway. SecA is the central component of the
Sec-dependent secretion pathway,[3e,6] interacting with virtually
all the other components of the system, acting as a molecular
chaperone and motor, and providing the driving force for pro-
tein translocation.[7] In addition, SecA has been shown to form
a protein-conducting channel in the presence of phospholipid
bilayers.[8] SecA is required for bacterial viability and virulence,
and is highly conserved in bacteria with no human counter-
part.[9] Targeting SecA therefore represents a unique strategy
to combat bacterial pathogens with minimal human toxicity. In
our previous studies, pyrimidine analogues were developed
from virtual screening against an Escherichia coli (Ec)SecA crys-
tal structure.[10] By screening related compounds, we identified
one bistriazole compound (1, SCA-21, Figure 1), which inhibits
both the intrinsic ATPase activity and translocation ATPase ac-
tivity of EcSecA, with an IC50 value of ~30 mm. By dissecting
this hit, we designed and synthesized roughly 40 analogues,
among which some (such as 7b and 12a) were found to po-
tently inhibit SecA-dependent activities at high-nanomolar to
low-micromolar concentrations in an in vitro (vesicle) protein
translocation study and an ion channel oocyte assay. The inhib-
ition of these functional activities correlates with the inhibition
of bacterial growth.
Results and Discussion
[a] J. Cui,+ A. S. Chaudhary, C. Dai, K. Damera, W. Chen, B. Wang
Department of Chemistry
Chemistry
Georgia State University, Atlanta, GA 30303 (USA)
SCA-21 has a pyrimidine core conjugated to two substituted
triazoles in a symmetric fashion by a thioether bond. Notably,
these triazoles are 1,2,4-triazoles, which are different from the
1,2,3-triazoles synthesized via [2+3] cycloaddition reaction be-
tween an organic azido compound and an alkyne. To improve
its potency and understand its structure–activity relationships
(SAR), we started to simplify the structure by dissecting the hit
compound in half and removing part A. Figure 1 shows the
E-mail: wang@gsu.edu
[b] J. Jin,+ Y.-h. Hsieh, H. Zhang, P. C. Tai
Department of Biology, Center for Biotechnology and Drug Design and
Center for Diagnostics and Therapeutics
Georgia State University, Atlanta, GA 30303 (USA)
E-mail: biopct@gsu.edu
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500447.
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Figure 1. Optimization of the hit compound SCA-21.
general strategy of analogue design. Specifically, the plan was
to examine substitution effects in scaffold I, positional effects
in scaffold IIA, the importance of the triazole ring in scaffold
IIB, and the effect of conformational constraints around the tri-
azole and pyrimidine axis in scaffold III.
We were interested in modifying scaffold I by changing six
different groups (R1–R5 and X; Figure 1). Synthesis started by
reacting commercially available benzoyl chloride 2 with hydra-
zine carboamide 3, followed by self-condensation of 4 in the
presence of 5% sodium hydroxide under reflux conditions to
yield 5 (Scheme 1).[11]
The triazole-pyrimidine analogues 7, 9, 12, and 15 were
then synthesized by reacting key intermediate 5 under weakly
basic conditions with the appropriately substituted pyrimidines
(Schemes 2 and 3). Series 7 has a methylthio ether group at
the 2-position, whereas series 9 does not. In series 12, the
methylthio ether was replaced by either a bulkier thioether or
an oxoether.
Scheme 3 describes the synthesis of compound 15, which
contains scaffold I, but with an oxadiazole instead of a triazole
linking the pyrimidine and phenyl rings. Compound 15 was
synthesized by first reacting commercially available 3,5-bis(tri-
fluoromethyl)benzhydrazide (13) with carbon disulfide to give
14.[12] Subsequent reaction of 14 with 4,6-dichloro-2-(methyl-
thio)pyrimidine (6b), under weakly basic conditions, gave com-
pound 15.[13]
Analogues with scaffold II (Figure 1) were designed to evalu-
ate the effect of regioisomerism, that is, whether the given
substitution is at positions 2 or 4 of the pyrimidine ring (com-
pounds 16–18) as well as the importance of the triazole ring
(compound 20). Scheme 4 illustrates the synthesis of com-
pounds 16–18 and 20. Specifically, they were synthesized by
reaction of intermediates 5, 14, or 19 with 2,4,6-trichloro pyri-
midine compounds under weakly basic conditions at room
temperature.[12]
Analogues of scaffold class III (Figure 1) were designed to
examine the importance of conformational constraints, espe-
Scheme 1. Reagents and conditions: a) THF, 08C!RT, overnight; b) 5% NaOH, reflux, 5 h, 65–87% overall yields.
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Scheme 2. Reagents and conditions: a) K2CO3, acetone, RT, 2–3 h, 35–80%.
Scheme 3. Reagents and conditions: a) CS2, H2O/EtOH, reflux, 5 h, 65%; b) K2CO3, acetone, RT, 2–3 h, 80%.
Scheme 4. Reagents and conditions: a) K2CO3, acetone, RT, 2–3 h, 35–80%.
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Scheme 5. Reagents and conditions: a) CS2, NH2NH2, KOH/EtOH, 82%; b) DMF, 608C, 4 h, 76%; c) NaBH4 (1 equiv), EtOH, RT, 2 h, 82%; d) NaBH4 (10 equiv),
EtOH, RT, 2 h, 40%.
Figure 2. Screening for inhibition of the ATPase activity of EcSecAN68: The initial screening assays were carried out at 50 mm for all compounds.
cially around the triazole and pyrimidine axis. Compound 22
was synthesized from commercially available 3,5-bis(trifluoro-
methyl)benzhydrazide 21 by reaction with carbon disulfide
under strongly basic conditions (Scheme 5).[12] Then reaction of
22 with 4,6-dichloro-2-(methylthio)pyrimidine-5-carbaldehyde
(23) gave compound 24. Compounds 25 and 26 were synthe-
sized by reduction of 24 with varying equivalents of sodium
borohydride at room temperature.[14]
used antimicrobial assays as a gatekeeper to ensure that we
examine all compounds with potent antimicrobial activities, re-
gardless of the results from the EcSecAN68 assay. We used two
representative strains for antimicrobial assays: a Gram-positive
strain (S. aureus 6538) and a Gram-negative strain (E. coli
NR698),[4c,10b,17] the latter of which has an outer-membrane mu-
tation resulting in increased drug permeability. Initial screening
was conducted at a test compound concentration of 50 mm
(Figure 2). There were 22 compounds that showed greater
than 50% inhibition at this concentration. These compounds
were then evaluated further at various concentrations to allow
the determination of IC50 values (Tables 1–3). Two class I com-
pounds, 7b and 12a, showed significant enzyme inhibitory ac-
tivities at low-micromolar concentrations. Notably, the ana-
logue with the pyrimidine ring removed, 5a, showed lower ac-
tivity. Such results suggest that both the triazole and pyrimi-
dine rings are very important for inhibitory activity. Changing
the position of the triazole ring substituent to the 2-position
(class II, Figure 1, Table 2) decreases the inhibitory activity; con-
formationally constrained analogues (class III, Figure 1, Table 3)
also did not yield active compounds.
Biological evaluation
We first evaluated the inhibitory effect against the intrinsic
ATPase activity of EcSecAN68, which is a truncated form of
E. coli SecA that lacks the inhibitory/regulatory C terminus.[10b,15]
In previous publications, we discussed in detail the advantages
and disadvantages of various assays in screening for SecA in-
hibitory activities, and the need to use more than one assay
for validations.[4c,16] Briefly, we recognize that this is not an
ideal assay because EcSecAN68 may very well have a different
conformation from that of the membrane-bound form in live
bacteria, and the E. coli form may not represent the SecA from
both Gram-positive and Gram-negative species. However, this
assay allows rapid screening of a large number of compounds,
and all the other assays are of low throughput.[10b,15] We also
For class I compounds, adding a chloro group as the R4 sub-
stituent significantly increased inhibitory effect (7b, Table 1).
Replacing one or both trifluoromethyl R1 and R2 groups with
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Table 1. SAR studies for class I compounds.
Compd
SCA
R1
R2
R3
X
R4
R5
Y
IC50 [mm][a]
EcSecAN68
MIC [mm][b]
E. coli NR698
S. aureus 6538
1
5a
7a
7b
7c
7d
7e
7 f
7g
7h
7i
SCA-21
N/A
-CF3
-CF3
-CF3
-CF3
-CH3
-CH3
-F
N/A
-CF3
-CF3
-CF3
-CF3
-CH3
-CH3
-H
N/A
-H
-H
-H
-H
-H
-H
-H
-H
-H
-CF3
-H
-H
-H
-H
-H
-H
-H
N/A
-SH
-S-
-S-
-S-
-S-
-S-
-S-
-S-
-S-
-S-
-S-
-S-
-S-
-S-
-S-
-S-
-S-
-S-
-S-
-S-
-S-
-S-
-S-
-S-
-S-
N/A
N/A
-CH3
-Cl
-H
N/A
N/A
N/A
-NH-
-NH-
-NH-
-NH-
-NH-
-NH-
-NH-
-NH-
-NH-
-NH-
-NH-
-NH-
-NH-
-NH-
-NH-
-NH-
-NH-
-NH-
-NH-
-NH-
-NH-
-NH-
-NH-
-NH-
-O-
25.0
60.0
25.0
30.0
95.0
25.0
>250.0
33.3
4.7
50.0
>250.0
50.0
200.0
50.0
100.0
25.0
>250.0
>250.0
35.4
3.1
>250
8.3
12.5
>250.0
18.8
3.1
62.5
>250.0
50.0
175.0
41.6
>250.0
14.6
>250.0
>250.0
18.8
SCA-126
SCA-128
SCA-107
SCA-123
SCA-117
SCA-106
SCA-153
SCA-155
SCA-159
SCA-161
SCA-157
SCA-111
SCA-110
SCA-112
SCA-113
SCA-114
SCA-130
SCA-138
SCA-156
SCA-158
SCA-160
SCA-162
SCA-163
SCA-154
SCA-133
-SCH3
-SCH3
-SCH3
-SCH3
-SCH3
-SCH3
-SCH3
-SCH3
-SCH3
-SCH3
-H
-H
-SPh
-SPh
-OPh
-S(CH2)4CH3
-S(CH2)4OH
-SPh
-SPh
-SPh
-H
65.0
30.0
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
<50.0
>100.0
<50.0
<50.0
<50.0
>200.0
100.0
20.0
50.0
9.0
50.0
25.0
<50.0
<50.0
<50.0
<50.0
<50.0
<50
-F
-OCH3
-H
-F
-OCH3
-H
7j
9a
-CF3
-CH3
-CF3
-CF3
-CH3
-CF3
-CF3
-CF3
-F
-CF3
-OCH3
-H
-OCH3
-F
-H
-CH3
-CF3
-CF3
-CH3
-CF3
-CF3
-CF3
-F
-H
-OCH3
-H
-H
-H
9b
12a
12b
12c
12d
12e
12 f
12g
12h
12i
12j
12k
15
1.8
12.5
3.1
1.6
25.0
20.8
18.8
6.3
43.8
31.3
18.8
-H
-H
-H
-H
-CF3
-OH
-H
>250
12.5
>250
83.3
ꢀ100.0
-SPh
-SPh
-SPh
-SCH3
8.3
ꢀ250.0
175.0
>250.0
-CF3
-CF3
-H
200
>100
[a] Compound concentration required to inhibit EcSecAN68 ATPase activity by 50%. [b] Lowest compound concentration that inhibits bacterial growth
after 24 h incubation at 378C; E. coli NR698 (with an outer membrane mutation resulting in increased drug permeability) and S. aureus 6538, representing
Gram-negative and Gram-positive bacteria to screen all compounds.
Table 2. SAR tests for class IIA compounds.
Compd
SCA
R1
R2
Y
X
R3
IC50 [mm][a]
EcSecAN68
MIC [mm][b]
E. coli NR698
S. aureus 6538
16a
16b
16c
16d
16e
17
SCA-124
SCA-116
SCA-139
SCA-152
SCA-131
SCA-135
-CF3
-CH3
-CF3
-H
-CF3
-CF3
-CF3
-CH3
-CF3
-F
-CF3
-CF3
-NH-
-NH-
-NH-
-NH-
-NH-
-O-
-S-
-S-
-O-
-S-
-S-
-S-
-Cl
-Cl
-Cl
-Cl
-NHCH2Ph
-Cl
50.0
>50.0
<50.0
>100.0
>50.0
70.0
12.5
>164.0
250.0
200.0
ꢀ250.0
>250.0
6.3
100.0
37.5
ꢀ100.0
50.0
18.8
18
SCA-140
-CF3
-CF3
-NH-
-O-
<50.0
>250.0
250.0
[a] Compound concentration required to inhibit EcSecAN68 ATPase activity by 50%. [b] Lowest compound concentration that inhibits bacterial growth
after 24 h incubation at 378C.
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Table 3. SAR tests for class IIB and III compounds.
Compd
SCA
R1
Y
R2
IC50 [mm][a]
EcSecAN68
MIC [mm][b]
E. coli NR698
S. aureus 6538
20a
20b
24
25
26
SCA-127
SCA-137
SCA-174
SCA-177
SCA-175
-Br
-Et
N/A
N/A
N/A
N/A
N/A
-CH=N-
-CH2NH-
-CH2NH-
N/A
N/A
-Cl
-Cl
-H
<50.0
<50.0
<25.0
46.0
>250.0
>250.0
12.5
>250.0
>100.0
>250.0
>250.0
3.1
>100.0
>100.0
50.0
[a] Compound concentration required to inhibit EcSecAN68 ATPase activity by 50%. [b] Lowest compound concentration that inhibits bacterial growth
after 24 h incubation at 378C.
any other substituent (compounds 7 f–7i) significant-
Table 4. Inhibition of various SecA ATPase and in vitro protein translocation activities
of EcSecA.
ly decreased the bacteriostatic effect, suggesting that
the two trifluoromethyl groups are important for in-
hibition. Changing triazole to oxadiazole in the struc-
ture decreased inhibition greatly, suggesting that the
triazole group is very important (compound 15). Re-
placing the methylthio group with hydrogen de-
creased inhibitory capacity (compound 9b), yet the
methylthio group could be replaced with a phenylthio
(12a) or phenoxy group (12c) without significantly
changing the inhibitory effects.
Compd
IC50 [mm]
ATPase activity
EcSecAN68 EcSecA[a] EcSecATn[b]
Translocation activity
Membrane[c] SecA–liposomes[d]
1
7b
12a
18.0
30.0
20.0
32.0
>200.0
>200.0
20.0
28.0
ND
7.0
1.5
2.0
ND
2.0
4.8
[a] Intrinsic EcSecA ATPase activity is that of full EcSecA without membrane and
proOmpA. [b] Translocation ATPase activity is that of EcSecA in the presence of urea-
washed E. coli BA13 membrane) and proOmpA. [c] In vitro translocation activity with
membrane was determined by using OmpA-depleted and urea-washed 773 mem-
branes and immunoblots to determine the translocation efficiency of proOmpA. [d] In
vitro translocation activity with SecA–liposomes was determined by using reconstitut-
ed SecA-only liposomes and immunoblots to determine the translocation efficiency of
proOmpA.
Among all the compounds, 7b (SCA-107) and 12a
(SCA-112) are the two most potent analogues devel-
oped from this study. We therefore focused on these
two compounds, together with the original lead
compound 1, for further evaluation. As discussed in
previous studies,[4c,15] monitoring the inhibitory activi-
ty toward the soluble form of EcSecAN68 is a good
initial screen. However, gauging inhibitory potency in the
membrane lipid environment by using either an ion current
channel activity assay or protein translocation assay seems to
correlate more closely with antimicrobial activity.[8] We there-
fore subjected compounds 1, 7b, and 12a to such assays, and
found that the IC50 values for 7b and 12a were ~1.5–2.0 mm in
the membrane protein translocation assay using EcSecA and
2.0–4.8 mm in the SecA–liposomes translocation assay (Table 4).
We also examined these SecA inhibitors using the channel ac-
tivity assay in oocytes[8,18] (Table 5). These compounds showed
very effective inhibition of the ion channel activity of EcSecA,
with IC50 values of ~1.3–2.4 mm. We also examined the effects
of these compounds on the ion channel activity of SecA puri-
fied from other bacterial strains and then reconstituted with
liposomes or cognate membranes. Table 5 shows that these
compounds are also very effective against SecA from both
Gram-positive and Gram-negative bacteria, with IC50 values
ranging from 0.7 to 2.6 mm. The inhibitory potencies in the ion
Table 5. Inhibition of ion channel activity of various SecA–liposomes in
oocytes.
Compd
IC50 [mm][a]
BaSecA1
EcSecA
SaSecA1
BsSecA
SpSecA
1
7b
12a
2.4
1.6
1.3
1.6
0.6
1.0
1.5
0.7
1.0
2.6
2.1
2.3
1.0
0.7
1.3
[a] Ion channel activity of SecA-only liposomes was determined by
adding reconstituted SecA–liposome complex into oocytes and recording
the current induced by proOmpA; EcSecA: E. coli SecA, SaSecA1: S. aureus
SecA1, BaSecA1: B. anthracis SecA1, BsSecA: B. subtilis SecA, SpSecA:
Streptococcus pyogenes SecA.
channel and protein translocation assays correlate very well
with antimicrobial results (see Tables 1–4 and the section
below), demonstrating once again what we reported earlier,
that is: 1) the EcSecAN68 assay is appropriate for the initial
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rapid screening, and 2) the protein translocation and ion chan-
nel activity assays provide numerical values of inhibitory activi-
ty that more closely parallel the antimicrobial efficacy. Corollary
to these points is our examination of the effect of these three
inhibitors on full-length SecA in solution (Table 4). As discussed
previously,[15] the full-length Sec protein has an inhibitory/regu-
latory C terminus. In a non-membrane environment, the IC50
values of SecA inhibitors tend to be much higher than those
obtained from the EcSecAN68, channel activity, and protein
translocation assays. Therefore, the IC50 values obtained from
the full-length SecA assay in solution must be analyzed with
the effect of the inhibitory/regulatory C terminus taken into
consideration.
To better understand the mechanism of inhibition in view of
the difference on SecA ATPase activity and functional activities
with lipid/membranes, we conducted inhibition kinetics studies
of SecA–liposome channel activity using 7b (SCA-107) as a rep-
resentative. Figure 4 shows that 7b is a noncompetitive inhibi-
Thus far, the correlation between the inhibitory potency
data from the channel activity and protein translocation assays
and the antimicrobial data is consistent with the notion that
the antimicrobial results (Tables 1–5) are largely due to SecA in-
hibition. To further examine this correlation, we conducted
protein pull-down work. We synthesized a biotinylated ana-
logue of 7b (SCA-256), which was used to carry out the pro-
tein pull-down assay (Figure 3), the results of which showed
Figure 4. Ion channel activity against EcSecA and SaSecA1: The ion channel
activity of SecA–liposomes was determined by adding reconstituted SecA–
liposome complex into oocytes and recording the current induced by
proOmpA. EcSecA: E. coli SecA; SaSecA1: S. aureus SecA1.
tor of EcSecA and SaSecA. Such results support the idea that
the inhibitor does not bind directly in the ATP binding pocket.
Molecular modeling suggests that 7b partially blocks the entry
of the ATP binding site (Figure 5). To gain an initial understand-
ing of the possible binding site for these SecA inhibitors, dock-
ing studies were conducted with the SYBYL version 2.0 soft-
ware package. Figure 5A shows that SCA-107 resides at the in-
terface of monomers A and B, slightly toward the A monomer;
Figure 5B shows the individual amino acid residues flanking
SCA-107.
Figure 3. Validation of SaSecA1 as a drug target by pull-down assay:
a) Structure of SCA-256. b) Whole-cell lysate of S. aureus ATCC 6538 was
mixed with SCA-256 (biotinylated 7b analogue) for 1 h, then streptavidin
magnetic beads were used to pull out proteins that interact with SCA-256.
The interaction between SaSecA1 and SCA-256 was examined by western
blot with the cross-reacting EcSecA antibody (1:5000 dilution). The synthesis
of SCA-256 is described in the Supporting Information.
that SCA-256 maintains a reasonable level of inhibitory activity
toward EcSecAN68 ATPase (IC50 ꢁ60 mm). The western blot re-
sults show that SCA-256 can recognize and pull down Staphy-
lococcus aureus (Sa)SecA1 from whole-cell lysates (Figure 3B).
Such results demonstrated the specific interactions between
SecA and the inhibitors, further supporting the notion that
SaSecA1 is a target of this group of inhibitors. However, as
with any protein pull-down work, rarely is only one protein
pulled down. This is no exception for SCA-256. There are many
factors that could contribute to such results and still be consis-
tent with SecA being the key target for the antimicrobial ef-
fects of these inhibitors. Furthermore, it should be noted that
the channel activity and protein translocation with SecA-only
liposomes clearly showed the specific inhibition of SecA func-
tional activity.
As discussed above, SecA is a conserved protein in bacteria.
Thus there are good reasons to believe that SecA inhibitors
can function as broad-spectrum antimicrobials. Our SecA inhib-
ition data certainly support this notion. To examine this further,
we evaluated the inhibitory effects of 1 (SCA-21), 7b (SCA-
107), and 12a (SCA-112) against various strains of bacteria
(Table 6). The results show that these three inhibitors have ex-
cellent antimicrobial effects, with MIC values between 0.8 and
25 mm, which are also similar to results for the inhibition of
SecA-specific functions in the channel activity and protein
translocation assays (Tables 4 and 5), and further support the
idea that the antimicrobial target is SecA. In addition to bacter-
iostatic effects, hit compound 1 also exhibits bactericidal
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Figure 5. Molecular modeling of 7b (SCA-107) binding in EcSecA: A) Docking was performed with SYBYL 2.0 using PDB ID: 2FSG with SecA dimers A and B.
Compound 7b binds to the interface of the A and B monomers, closer to A (6 Š), partially blocking the entrance to the ATP site. B) SCA-107 (ball-and-stick
structure) surrounded by residues of the EcSecA active site. Red arrows indicate residues that are in SCA-107 vicinity but do not form bonds with the mole-
cule.
Table 6. Bacteriostatic effects against various bacterial strains.[a]
Compd
MIC [mm][b]
E. coli NR698 B. subtilis 168 B. anthracis Sterne S. aureus 6538 S. aureus Mu50 S. aureus N315 S. aureus Mu3 S. aureus Newman
1
7b
12a
25
5.2
3.1
6.3
1.6
0.8
6.3
3.1
1.6
12.5
3.1
1.8
ND
1.6
0.8
ND
0.8
0.8
ND
3.1
1.6
ND
1.6
ND
[a] Log-phase cells were diluted to OD600 ꢁ0.05, followed by incubation with varying concentrations of compounds for 16 h at 378C. [b] Minimal com-
pound concentration required to inhibit cell growth.
effects, with generally more than 2 log units decrease in CFU
values at 25 mm against some S. aureus strains, as listed in
Table 7. Compound 7b decreased the CFU count of S. aureus
Mu50 by 2 log units at 50 mm (Figure 6).
Some methicillin-resistant S. aureus (MRSA) strains acquire
multidrug resistance in addition to their resistance to methicil-
lin. We further examined the efficacy of our inhibitors against
one strain, Mu50, in comparison with antibiotics in clinical use.
The results show that 7b (SCA-107, MIC: 0.75 mgmLÀ1) and
12a (SCA-112, MIC: 0.43 mgmLÀ1) are more effective than most
clinically used antibiotics including ampicillin (1000 mgmLÀ1
>1300-fold), polymyxin B (31 mgmLÀ1, >40-fold), erythromycin
(1250 mgmLÀ1 >1600-fold), tetracycline (63 mgmLÀ1
>80-
fold), kanamycin (1000 mgmLÀ1, >1300-fold), rifampicin
(1000 mgmLÀ1, >1300-fold), norfloxacin (250 mgmLÀ1, >330-
fold), and even vancomycin (8 mgmLÀ1, 10-fold for 7b, 18-fold
for 12a), the latter of which is considered as the last-resort
drug for treating many drug-resistant bacterial infections.
These SecA inhibitors also exert bactericidal effects on a wide
range of bacteria, including S. aureus Mu50, S. aureus N6538,
,
,
,
Table 7. Antimicrobial activities against S. aureus efflux strains.[a]
Compd
SCA
WT 8325-4
NorAÀK1758
NorA+ +K2361
MepAÀK2908
MepA+ +K2068
1[b]
7b
SCA-21
SCA-107
SCA-112
8
2.1
1.6
8
3.1
1.6
8
3.1
1.8
8
2.1
1.0
6
3.1
1.3
Bacteriostatic
MIC [mm]
12a
Bactericidal effect
log[decrease CFU]
1
SCA-21
3
3
4
2
3
[a] Deletion (NorAÀ, MepAÀ) and overexpression (NorA+ +, MepA+ +) of NorA and MepA efflux pump strains, K1758, K2361, K2908, and K2068 are deriva-
tives from S. aureus 8325-4. Bactericidal effects were determined by counting CFU after 1 h treatment at 378C with compounds at 25 mm concentration.
[b] The initial MIC tested for compound 1 was from the tube assay.
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of SecA is the fact that it is a widely conserved membrane pro-
tein, responsible for the secretion of virulence factors, and di-
rectly accessible from the extracellular matrix without the need
for certain intracellular concentrations. All these combined sug-
gest that SecA inhibitors have the potential for being devel-
oped as broad-spectrum antimicrobials, can attenuate the
pathogenicity of bacteria by directly inhibiting virulence factor
production, and can overcome the effect of efflux pumps,
which are responsible for multidrug resistance.
Figure 6. Bactericidal effect of 7b (SCA-107) against S. aureus Mu50: Bacteri-
cidal effects were determined by counting CFU after 2 h treatment with vari-
ous concentrations of 7b.
Experimental Section
Chemistry
All reagents were purchased from Acros or Aldrich and were used
as received. 1H and 13C NMR spectra were collected on
a Bruker 400 NMR spectrometer. Mass spectrometric analyses were
performed by the Mass Spectrometry facilities at Georgia State Uni-
versity. Molecular modeling was conducted with SYBYL-X 2.0, and
the standard procedures provided in the manual were used. Spe-
cifically, Surflex-dock was used to dock ligands into the SecA crys-
tal structure (PDB ID: 2FSG) using automatic docking. All standard
parameters provided in SYBYL-X 2.0 were used for energy minimi-
zation of the crystal structure of SecA. The AMBER7 FF99 force
field, along with the default parameters described in the manual,
were used, and a maximum of 20 docking poses were generated
for each molecule.
and B. anthraces Sterne (Table 6, and data not shown). We pre-
viously described two other classes of structurally different
SecA inhibitors,[16b,17] and this novel class is the most effective
in terms of both bacteriostatic and bactericidal activity.
Compared with other known antimicrobial targets, one of
the unique features of SecA is that it functions as a membrane
protein. Therefore, inhibitors should be able to access SecA
without the need for intracellular accumulation. One conse-
quence of this ready accessibility of SecA is that efflux pumps
are not expected to affect the potency of SecA inhibitors. To
assess this, we examined various MRSA strains, which are
known to possess multidrug-resistance efflux pumps and thus
render many drugs ineffective. The results showed that growth
inhibition by these SecA inhibitors is independent of the level
of efflux pump expression in these strains (Table 7). Such re-
sults are consistent with the notion that SecA may be accessi-
ble by this class of inhibitors from the extracellular matrix.
SecA inhibitors may therefore exert their effect without the
need to enter the cell, and thus have the capacity to attenuate
the effect of efflux pumps, a major mechanism behind multi-
drug resistance.
2-(3,5-Bis(trifluoromethyl)benzoyl)hydrazinecarbothioamide
(4a): To a mixture of 3,5-bis(trifluoromethyl)benzoyl chloride (1 g,
3.63 mmol) in 25 mL tetrahydrofuran was added hydrazine carbo-
thioamide (0.73 g, 7.98 mmol) slowly at 0–58C. The temperature
was increased to room temperature, and the reaction was stirred
overnight. The reaction was then stopped with the addition of sa-
turated sodium carbonate, and the mixture was extracted with
EtOAc (3”50 mL). The combined organic layers were washed with
water and brine, and dried over Na2SO4. The solid was filtered off,
and the solvent was evaporated under reduced pressure to afford
a crude product, which was used directly for the next step.
All the evidence from inhibition of SecA-dependent protein
translocation, channel activity kinetic studies, and antimicrobial
studies indicate that these SecA inhibitors exert their antimi-
crobial effect by the inhibition of SecA functions in the mem-
branes, and possess the ability to bypass or minimize the
effects of efflux pumps.
5-(3,5-Bis(trifluoromethyl)phenyl)-4H-1,2,4-triazole-3-thiol (5a):
To the crude product 4a was added 5% sodium hydroxide solution
(50 mL), and the mixture was heated at reflux for 5 h. Then 2n HCl
was added to adjust the mixture to pH~6–7. The white solid was
filtered, washed with water 3–5 times, and then dried under
vacuum at room temperature to give 5a as a white powder
1
(423 mg, 65% overall for two steps). H NMR (DMSO): d=13.71 (s,
1H), 13.62 (s, 1H), 7.54 (s, 2H), 7.14 (s, 1H), 2.31 ppm (s, 6H);
13C NMR (DMSO): d=168.1, 148.3, 131.8, 131.4, 128.3, 126.5, 124.7,
124.3, 122.0 ppm; ESI-MS: 314.3 [M+H]+.
Conclusions
In summary, we have synthesized and evaluated a new class of
triazole-pyrimidine analogues as novel SecA inhibitors. Com-
pounds 7b (SCA-107) and 12a (SCA-112) showed the most
potent activities, with IC50 and MIC values in the low- to sub-
micromolar range. In addition, results from target identification
assays are consistent with SecA being a target. The inhibition
of SecA-dependent channel activity and protein translocation
correlates well with antimicrobial activity, further suggesting
that these compounds exert their antimicrobial effect through
SecA inhibition. However, this does not exclude the possibility
of off-target effects in the antimicrobial assay. A unique feature
5-(3,5-Bis(trifluoromethyl)phenyl)-4H-1,2,4-triazol-3-amine (5b):
Synthesis of 5b followed the same procedure as for 5a in 64%
1
yield as a white powder. H NMR (DMSO): d=8.38 (s, 2H), 8.06 (s,
1H), 6.33 (s, 2H); 13C NMR (DMSO): d=158.6, 156.1, 135.0, 131.3,
131.0, 125.5, 125.0, 122.3, 121.9 ppm; ESI-MS: 297.0 [M+H]+.
5-(3,5-Bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-3-ol (5c): Syn-
thesis of 5c followed the same procedure as for 5a in 56% yield
1
as a white powder. H NMR (DMSO): d=8.41 (s, 2H), 8.36 (s, 1H),
2.50 ppm (s, 2H); 13C NMR (DMSO): d=165.1, 134.6, 131.3, 131.1,
130.7, 129.9, 127.4, 126.7, 124.7, 121.9 ppm; ESI-MS: 298.0 [M+
H]+.
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5-(3-Fluorophenyl)-1H-1,2,4-triazole-3-thiol (5d): Synthesis of 5d
followed the same procedure as for 5a in 66% yield as a white
powder. 1H NMR (DMSO): d=13.92 (s, 1H), 13.77 (s, 1H), 7.35–
7.76 ppm (m, 4H); 13C NMR (DMSO): d=167.6, 163.9, 161.5, 149.5,
131.9, 128.0, 122.2, 118.0, 113.0 ppm; HRMS-ESI (+): calcd for
C8H7N3SF: 196.03, found: 196.03 [M+H]+.
60.2, 21.3, 14.1 ppm; HRMS-ESI (+): calcd for C15H14N5S2Cl:
364.0457, found: 364.0457 [M+H]+.
4-Chloro-6-((5-(3-fluorophenyl)-4H-1,2,4-triazol-3-yl)thio)-2-
(methylthio)pyrimidine (7 f): Synthesis of 7 f followed the same
procedure as for 7a in 46% yield as a white off solid. 1H NMR
(DMSO): d=7.87–7.85 (d, J=7.6 Hz, 1H), 7.78–7.76 (d, J=9.2 Hz,
1H), 7.62–7.56 (dd, J=7.6, 5.6 Hz, 1H), 7.37–7.33 (t, J=7.6 Hz, 1H),
7.23 (s, 1H), 2.50 (s, 3H), 2.34 ppm (s, 3H); 13C NMR (DMSO): d=
172.6, 170.8, 164.0, 161.5, 160.0, 131.9, 131.8, 122.7, 117.8, 117.6,
113.4, 113.1, 112.8, 14.04 ppm; ESI-MS (+): 354.0 [M+H]+.
5-(3,5-Difluorophenyl)-1H-1,2,4-triazole-3-thiol (5e): Synthesis of
5e followed the same procedure as for 5a in 67% yield as a white
powder. 1H NMR (DMSO): d=8.34 (s, 2H), 8.32 ppm (s, 1H);
13C NMR (DMSO): d=131.9, 131.6, 131.5, 127.6, 125.0, 124.6,
121.9 ppm.
4-Chloro-6-((5-(3,5-difluorophenyl)-4H-1,2,4-triazol-3-yl)thio)-2-
(methylthio)pyrimidine (7g): Synthesis of 7g followed the same
5-(4-(Trifluoromethyl)phenyl)-1H-1,2,4-triazole-3-thiol (5h): Syn-
thesis of 5h followed the same procedure as for 5a in 72% yield
1
procedure as for 7a in 47% yield as a white solid. H NMR (DMSO):
1
d=7.67–7.65 (m, 1H), 7.44 (m, 1H), 7.26 (s, 1H), 2.33 ppm (S, 3H).
as a white powder. H NMR (DMSO): d=7.54 (s, 2H), 7.14 (s, 1H),
2.31 ppm (s, 6H); 13C NMR (DMSO): d=167.3, 150.8, 138.7, 132.4,
4-Chloro-6-((5-(3,5-dimethoxyphenyl)-4H-1,2,4-triazol-3-yl)thio)-
2-(methylthio)pyrimidine (7h): Synthesis of 7h followed the same
procedure as for 7a in 59% yield as a brown solid. 1H NMR
(DMSO): d=15.01(s, 1H), 7.16 (s, 3H), 6.61 (s, 1H), 3.80 (s, 6H),
2.49 ppm (s, 3H); 13C NMR (DMSO): d=172.5, 161.3, 160.0, 104.4,
102.9, 60.2, 55.8, 14.5 ppm; ESI-MS (+): 396.03 [M+H]+.
125.7, 123.8 ppm.
4-((5-(3,5-Bis(trifluoromethyl)phenyl)-4H-1,2,4-triazol-3-yl)thio)-
6-methyl-2-(methylthio)pyrimidine (7a): To compound 5a
(70 mg, 0.34 mmol) in 10 mL acetone was added potassium car-
bonate (65.2 mg, 0.47 mmol) and 4,6-dichloro-2-(methylthio)pyrimi-
dine (6a, 36.3 mg, 0.2 mmol). The reaction mixture was stirred at
room temperature overnight. Then the reaction was stopped with
the addition of 4n HCl, and extracted with EtOAc (3”30 mL). The
combined organic layers were washed with water and brine, dried
over Na2SO4, and filtered. After purification by silica gel column
chromatography (hexane/EtOAc 10:1), 7a (79 mg, 64%) was ob-
tained as a white solid. 1H NMR (DMSO): d=8.60 (s, 2H), 8.29 (s,
1H), 7.96 (s, 1H), 6.95 (s, 1H); 2.32 ppm (s, 6H); 13C NMR (DMSO):
d=171.4, 167.7, 162.7, 131.9, 131.6, 126.7, 124.8, 123.7, 122.1,
113.1 ppm; MS-ESI (+): calcd for C16H11F6N5S2: 452.41, found:
452.04 [M+H]+.
4-Chloro-2-(methylthio)-6-((5-(4-(trifluoromethyl)phenyl)-4H-
1,2,4-triazol-3-yl)thio)pyrimidine (7i): Synthesis of 7i followed the
1
same procedure as for 7a in 52% yield as a white powder. H NMR
(DMSO): d=8.22–8.20 (d, J=8.0 Hz, 2H), 7.90-7.88 (d, J=8.0 Hz,
2H), 7.26 (s, 1H), 2.32 ppm (s, 3H); 13C NMR (DMSO): d=172.6,
170.4, 160.0, 130.8, 130.5, 128.8, 126.5, 126.4, 123.3, 112.9,
14.9 ppm.
4-Chloro-2-(methylthio)-6-((5-(3-(trifluoromethyl)phenyl)-4H-
1,2,4-triazol-3-yl)thio)pyrimidine (7j): Synthesis of 7j followed the
1
same procedure as for 7a in 39% yield as a white solid. H NMR
(DMSO): d=8.46–7.85 (m, 5H), 2.51 ppm (s, 3H).
4-((5-(3,5-Bis(trifluoromethyl)phenyl)-4H-1,2,4-triazol-3-yl)thio)-
6-chloro-2-(methylthio)pyrimidine (7b): Synthesis of 7b followed
the same procedure as for 7a in 67% yield as a light brown solid.
1H NMR (DMSO): d=15.50 (brs, 1H), 8.57 (s, 2H), 8.25 (s, 1H), 7.28
(s, 1H), 2.34 ppm (s, 3H); 13C NMR (DMSO): d=172.7, 160.1, 131.8,
131.5, 131.2, 126.8, 124.8, 122.1, 113.0, 14.0 ppm; HRMS-ESI (+):
calcd for C15H8N5S2F6Cl: 471.9874, found: 471.9856 [M+H]+.
4-Chloro-6-((5-(3,5-dimethylphenyl)-4H-1,2,4-triazol-3-yl)thio)-
pyrimidine (9a): Synthesis of 9a followed the same procedure as
for 7a in 57% yield as a white solid. 1H NMR (DMSO): d=15.07
(brs, 1H), 8.84 (s, 1H), 7.66 (s, 2H), 7.53 (s, 1H), 7.18 (s, 1H),
2.35 ppm (s, 6H); 13C NMR (DMSO): d=160.4, 158.7, 138.86, 124.4,
118.0, 21.3 ppm; ESI-MS (+): 318.0 [M+H]+.
4-((5-(3,5-Bis(trifluoromethyl)phenyl)-4H-1,2,4-triazol-3-yl)thio)-
6-chloropyrimidine (9b): Synthesis of 9b followed the same pro-
cedure as for 7a in 47% yield as a white solid. 1H NMR (DMSO):
d=15.52 (brs, 1H), 8.85 (s, 1H), 8.61 (s, 2H), 8.31 (s, 1H), 7.66 ppm
(s, 1H); 13C NMR (DMSO): d=160.6, 158.8, 132.1, 131.8, 131.5,
131.1, 126.8, 124.8, 124.0, 122.1, 118.3 ppm; ESI-MS: 426.0 [M+H]+
.
4-((5-(3,5-Bis(trifluoromethyl)phenyl)-4H-1,2,4-triazol-3-yl)thio)-
2-(methylthio)pyrimidine (7c): Synthesis of 7c followed the same
procedure as for 7a in 62% yield as a white solid. H NMR (CDCl3):
d=13.34 (brs, 1H), 8.60 (s, 2H), 8.39 (d, J=5.6 Hz, 1H), 7.93 (s,
1H), 7.01 (d, J=5.6 Hz, 1H), 2.57 ppm (s, 3H); 13C NMR (CDCl3): d=
173.4, 164.7, 160.1, 156.2, 132.4, 132.3, 132.0, 126.5, 124.5, 123.1,
121.8, 114.0, 14.2 ppm; ESI-MS: 438.0 [M+H]+; HRMS-ESI (+): calcd
for C15H10F6N5S2: 438.0282, found: 438.0280 [M+H]+.
1
4,6-Dichloro-2-(phenylthio)pyrimidine (11a): To a mixture of thio-
phenol (0.28 mL, 2.73 mmol), potassium carbonate (565 mg,
4.1 mmol) and 5 mL acetone in a 10 mL round-bottom flask was
added 10 and 2,4,6-trichloropyrimidine (500 mg, 2.73 mmol) at
room temperature. The mixture was kept stirring overnight. Then
the solid was filtered off, and the reaction mixture was concentrat-
ed by evaporation under vacuum. The residue was purified by
silica gel column chromatography (hexane/EtOAc 25:1) to give 11 a
(600 mg), which was used directly in the next step without purifi-
cation.
4-((5-(3,5-Dimethylphenyl)-4H-1,2,4-triazol-3-yl)thio)-2-(methyl-
thio)pyrimidine (7d): Synthesis of 7d followed the same proce-
1
dure as for 7a in 57% yield as a white solid. H NMR (DMSO): d=
8.78 (d, J=5.2 Hz, 1H), 8.60 (d, J=5.2 Hz, 1H), 7.92 (d, J=5.2 Hz,
1H), 7.62 (m, 2H), 7.11 (s, 1H), 2.61 (s, 3H), 2.34 ppm (s, 6H);
13C NMR (DMSO): d=166.1, 160.5, 157.9, 138.4, 132.4, 124.4, 116.9,
21.3, 14.2, 13.9 ppm; ESI-MS (+): 330.1 [M+H]+; HRMS-ESI (+):
calcd for C15H16N5S2: 330.0847, found: 330.0841 [M+H]+.
4-Chloro-6-((5-(3,5-dimethylphenyl)-4H-1,2,4-triazol-3-yl)thio)-2-
(methylthio)pyrimidine (7e): Synthesis of 7e followed the same
procedure as for 7a in 76% yield as a white solid. H NMR (DMSO):
d=15.06 (brs, 1H), 7.64 (s, 2H), 7.15 (s, 2H), 2.37 (s, 3H), 2.34 ppm
(s, 6H); 13C NMR (DMSO): d=172.6, 160.0, 138.8, 132.5, 124.4, 112.7,
4-((5-(3,5-Bis(trifluoromethyl)phenyl)-4H-1,2,4-triazol-3-yl)thio)-
6-chloro-2-(phenylthio)pyrimidine (12a): To
a mixture of 5a
1
(470 mg, 1.5 mmol), potassium carbonate (276 mg, 2 mmol) and
acetone (5 mL) in a 10 mL round-bottom flask, was added 11 a
(300 mg) at room temperature. The reaction was kept stirring at
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room temperature overnight. Then the solid portion was filtered
off, and the residue after solvent evaporation was purified by silica
gel column chromatography (hexane/EtOAc 25:1!10:1) to give
4-Chloro-2-(phenylthio)-6-((5-(4-(trifluoromethyl)phenyl)-4H-
1,2,4-triazol-3-yl)thio)pyrimidine (12i): Synthesis of 12i followed
the same procedure as for 12a in 59% yield as a white solid.
1H NMR (DMSO): d=8.22–8.20 (d, J=8.0 Hz, 2H), 7.90–7.88 (d, J=
8.0 Hz, 2H), 7.26 (s, H), 2.32 ppm (s, 3H); 13C NMR (DMSO): d=
172.6, 170.4, 160.0, 130.8, 130.5, 128.8, 128.4, 125.5, 123.0, 111.8,
14.0 ppm.
1
12a as a white solid (478 mg, 51% for two steps). H NMR (DMSO):
d=15.34 (brs, 1H), 8.58 (s, 2H), 8.29 (s, 1H), 7.44 (m, 3H), 7.20 (m,
2H), 7.11 ppm (m, 1H); 13C NMR (DMSO): d=171.9, 160.2, 135.1,
132.1, 131.8, 131.5, 129.7, 127.7, 126.8, 124.9, 124.0, 122.2,
114.1 ppm; ESI-MS (+): 534.0 [M+H]+.
3-(5-((6-Chloro-2-(phenylthio)pyrimidin-4-yl)thio)-4H-1,2,4-tri-
azol-3-yl)-5-methoxy phenol (12j): Synthesis of 12j followed the
same procedure as for 12a in yield 31% yield as a white solid.
1H NMR (DMSO): d=7.47–7.45 (d, J=7.2 Hz, 2H), 7.21–7.19 (d, J=
7.2 Hz, 2H), 7.11–7.09 (d, J=7.2 Hz, 1H), 6.95 (s, 2H), 6.54 (s, 1H),
6.07 (s, 1H), 3.81 ppm (s, 3H); 13C NMR (DMSO): d=175.7, 160.3,
159.4, 158.6, 135.6, 131.9, 125.9, 110.8, 108.6, 104.1, 103.1,
58.8 ppm.
4-Chloro-6-((5-(3,5-dimethylphenyl)-4H-1,2,4-triazol-3-yl)thio)-2-
(phenylthio)pyrimidine (12b): Synthesis of 12b followed the
same procedure as for 12a in 56% yield as a white solid. H NMR
(CD3OD): d=7.56 (m, 2H), 7.38 (m, 2H), 7.17 (m, 5H), 2.40 ppm (s,
6H); 13C NMR (CD3OD): d=172.6, 170.8, 160.0, 138.8, 138.6, 134.8,
132.1, 129.1, 128.7, 127.8, 126.5, 123.9, 123.8, 112.8, 20.1 ppm; ESI-
MS (+): 426.1 [M+H]+.
1
4-((5-(3,5-Bis(trifluoromethyl)phenyl)-4H-1,2,4-triazol-3-yl)thio)-
6-chloro-2-phenoxypyrimidine (12c): Synthesis of 12c followed
the same procedure as for 12a in 43% yield as a white solid.
1H NMR (DMSO): d=15.52 (brs, 1H), 8.56 (m, 3H), 7.49 ppm (m,
7H); 13C NMR (DMSO): d=170.5, 163.5, 161.8, 158.9, 152.2, 151.9,
131.8, 131.4, 130.5, 129.8, 126.9, 125.8, 124.8, 124.1, 122.1, 121.8,
112.7, 107.7, 103.1 ppm; ESI-MS (+): 518.0 [M+H]+.
3-(5-((6-Chloro-2-(phenylthio)pyrimidin-4-yl)thio)-4H-1,2,4-tri-
azol-3-yl)-5-fluorophenol (12k): Synthesis of 12k followed the
same procedure as for 12a in 54% yield as a light brown solid.
1H NMR (CDCl3): d=7.68 (m, 1H), 7.62–7.60 (d, J=9.2 Hz, 1H),
7.43–7.39 (dd, J=9.2, 7.2 Hz, 3H), 7.23–7.13 (m, 4H), 6.32 ppm (s,
1H); 13C NMR (CDCl3): d=176.4, 169.3, 164.1, 159.2, 135.5, 130.7,
130.1, 126.1, 122.2, 122.1, 117.7, 117.4, 113.8, 113.6, 111.2 ppm;
HRMS-ESI (+): calcd for C18H11ClN5S2: 416.0208, found: 416.0207
[M+H]+.
4-((5-(3,5-Bis(trifluoromethyl)phenyl)-4H-1,2,4-triazol-3-yl)thio)-
6-chloro-2-(hexylthio)pyrimidine (12d): Synthesis of 12d followed
the same procedure as for 12a in 36% yield as a white solid.
1H NMR (CDCl3): d=8.61 (s, 2H), 7.94 (s, 1H), 7.03 (s, 1H), 3.15–3.11
(t, J=7.6, 7.2 Hz, 2H), 1.75–1.70 (m, 2H), 1.43–1.27 (m, 6H), 0.95–
0.92 ppm (m, 3H); 13C NMR (CDCl3): d=173.9, 165.7, 160.5, 132.4,
132.1, 132.0, 126.4, 124.5, 123.1, 121.8, 117.8, 113.1, 31.5, 30.9, 30.8,
30.1, 28.3, 22.1, 13.8 ppm; ESI-MS: 528.1 [M+H]+.
5-(3,5-Bis(trifluoromethyl)phenyl)-1,3,4-oxadiazole-2-thiol (14):
Synthesis of 14 followed the same procedure as for 5a in 69%
1
yield as a white solid. H NMR (CDCl3): d=11.7 (s, 1H), 8.44 (s, 2H),
8.09 ppm (s, 1H); 13C NMR (CDCl3): d=178.1, 158.8, 133.0, 126.4,
125.8, 124.4, 123.8, 121.1 ppm; HRMS-ESI (+): calcd for
C10H4F6N2OS: 315.0021, found: 315.0027 [M+H]+.
4-((4-((5-(3,5-Bis(trifluoromethyl)phenyl)-4H-1,2,4-triazol-3-yl)-
thio)-6-chloropyrimidin-2-yl)thio)butan-1-ol (12e): Synthesis of
12e followed the same procedure as for 12a in 33% yield as
2-(3,5-Bis(trifluoromethyl)phenyl)-5-((6-chloro-2-(methylthio)pyr-
imidin-4-yl)thio)-1,3,4-oxadiazole (15): Synthesis of 15 followed
the same procedure as for 7a in 58% yield as a white solid.
1H NMR (CDCl3): d=8.57 (s, 2H), 8.12 (s, 1H), 7.21 (s, 1H), 2.35 ppm
(s, 3H); 13C NMR (CDCl3): d=174.2, 165.5, 165.1, 161.0, 158.3, 133.4,
133.1, 127.0, 125.9, 123.9, 121.2, 113.0, 14.2 ppm; ESI-MS: 472.8
[M+H]+; HRMS-ESI (+): calcd for C15H8ClF6N4OS2: 472.9719, found:
472.9722 [M+H]+.
1
a white solid. H NMR (CDCl3): d=8.59 (s, 2H), 7.94 (s, 1H), 6.96 (s,
1H), 3.76–3.73 (m, 2H), 2.94–2.90 (m, 2H), 1.73–1.62 ppm (m, 4H);
13C NMR (CDCl3): d=173.3, 167.1, 160.4, 132.5, 132.2, 131.9, 127.1,
124.4, 123.3, 121.7, 113.0, 62.2, 31.1, 30.8, 25.5 ppm; HRMS-ESI (+):
calcd for C18H14ClF6N5OS2: 530.0316, found: 530.0318 [M+H]+.
4-Chloro-6-((5-(3,5-difluorophenyl)-4H-1,2,4-triazol-3-yl)thio)-2-
(phenylthio)pyrimidine (12 f): Synthesis of 12 f followed the same
procedure as for 12a in 59% yield as a white solid. 1H NMR
(CDCl3): d=7.56–7.43 (m, 7H), 6.50 ppm (s, 2H); 13C NMR (CDCl3):
d=178.6, 164.5, 164.4, 162.3, 161.8, 158.3, 135.6, 131.0, 130.4,
126.0, 121.6, 108.6, 105.2 ppm.
2-((3-(3,5-Bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-5-yl)thio)-
4,6-dichloropyrimi-dine(16a): Synthesis of 16a followed the same
procedure as for 7a in 78% yield as a white solid. H NMR (CDCl3):
d=8.59 (s, 2H), 7.95 (s, 1H), 7.37 ppm (s, 1H); 13C NMR (CDCl3): d=
169.2, 162.1, 160.1, 132.5, 132.2, 131.8, 126.55, 124.4, 123.4, 121.7,
119.0, 116.7 ppm; ESI-MS: 461.9 [M+H]+; HRMS-ESI (+): calcd for
C14H5Cl2F6N5S: 459.9637, found: 459.9638 [M+H]+.
1
4-Chloro-2-(phenylthio)-6-((5-(3-(trifluoromethyl)phenyl)-4H-
1,2,4-triazol-3-yl)thio)pyrimidine (12g): Synthesis of 12g followed
the same procedure as for 12a in 41% yield as a white solid.
1H NMR (CDCl3): d=8.15–8.13 (d, J=7.6 Hz, 1H), 7.14–7.12 (d, J=
8.0 Hz, 1H), 7.72–7.59 (dd, J=7.6, 8.0 Hz, 1H), 7.64–7.44 (dd, J=
8.8, 7.2 Hz, 2H), 7.21–7.14 (m, 3H), 6.34 ppm (s, 1H); 13C NMR
(CDCl3): d=176.5, 159.2, 135.5, 131.5, 131.2, 130.6, 129.8, 128.8,
125.7, 123.6, 123.5, 122.4, 111.3 ppm.
4,6-Dichloro-2-((3-(3,5-dimethylphenyl)-1H-1,2,4-triazol-5-yl)th-
io)pyrimidine (16b): Synthesis of 16b followed the same proce-
1
dure as for 7a in 74% yield as a white solid. H NMR (DMSO): d=
15.1 (s, 1H), 7.64 (s, 2H), 7.52 (s, 1H), 7.17 (s, 1H), 2.35 ppm (s, 6H);
13C NMR (DMSO): d=174.8, 161.4, 158.9, 157.5, 151.3, 136.8, 132.6,
128.0, 124.5, 118.3, 21.3 ppm; ESI-MS: 352.1 [M+H]+; HRMS-ESI
(+): calcd for C14H12Cl2N5OS: 352.0184, found: 352.0190 [M+H]+.
4-Chloro-6-((5-(3,5-dimethoxyphenyl)-4H-1,2,4-triazol-3-yl)thio)-
2-(phenylthio)pyrimidine (12h): Synthesis of 12h followed the
same procedure as for 12a in 45% yield as a white solid. H NMR
(CDCl3): d=7.56–7.55 (d, J=4.4 Hz, 2H), 7.26 (m, 5H), 7.06 (s, 2H),
3.83 ppm (s, 6H); 13C NMR (CDCl3): d=176.1, 161.2, 159.2, 135.3,
130.6, 130.1, 126.3, 111.1, 104.4, 102.9, 55.6 ppm; HRMS-ESI (+):
calcd for C20H16ClN5O2S2: 458.0526, found: 458.0567 [M+H]+.
4,6-Dichloro-2-((3-(3,5-dimethylphenyl)-1H-1,2,4-triazol-5-yl)oxy)-
pyrimidine (16c): Synthesis of 16c followed the same procedure
1
1
as for 7a in 64% yield as a white solid. H NMR (CDCl3): d=8.56 (s,
2H), 8.11 (s, 1H), 7.66 ppm (s, 1H); 13C NMR (CDCl3): d=167.6,
165.6, 162.7, 160.4, 157.4, 133.4, 133.0, 127.1, 125.9, 124.9, 123.9,
121.2, 117.0 ppm; ESI-MS: 445.3 [M+H]+.
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4,6-Dichloro-2-((5-(3-fluorophenyl)-4H-1,2,4-triazol-3-yl)thio)pyri-
midine (16d): Synthesis of 16d followed the same procedure as
for 7a in 71% yield as a white solid. H NMR (CDCl3): d=7.77–7.75
(d, J=7.2 Hz, 1H), 7.70–7.68 (d, J=9.2 Hz, 1H), 7.45–7.39 (dd, J=
8.4, 7.2 Hz, 1H), 7.26 (s, 1H), 7.19–7.14 ppm (m, 1H); 13C NMR
(CDCl3): d=171.8, 164.1, 161.7, 130.9, 129.0, 122.2, 118.1, 117.8,
116.3, 113.8, 113.6 ppm; HRMS-ESI (+): calcd for C12H7Cl2FN5S:
341.9784, found: 341.9783 [M+H]+.
chloro-2-(methylthio)pyrimidine-5-carbaldehyde (223 mg, 1 mmol)
in DMF (6 mL) was heated at 608C for 4 h. After cooling to room
temperature, 20 mL water was added. The precipitate was filtered
off and the resulting solid was further purified by flash chromatog-
raphy (hexane/EtOAc 1:1) to afford 24 as a white solid (380 mg,
76%). 1H NMR (DMSO): d=8.88 (s, 1H), 8.50 (s, 2H), 8.38 (s, 1H),
2.63 ppm (s, 3H); HRMS (ESI) calcd for C16H8ClF6N6S2: 496.9839,
found: 496.9828 [M+H]+.
1
3-(3,5-Bis(trifluoromethyl)phenyl)-7-chloro-9-(methylthio)-5,6-di-
N-Benzyl-2-((3-(3,5-bis(trifluoromethyl)phenyl)-4H-1,2,4-triazol-5-
yl)thio)-6-chloro-pyrimidin-4-amine (16e): Synthesis of 16e fol-
lowed the same procedure as for 7a in 45% yield as a white solid.
1H NMR (CDCl3): d=7.55 (s, 3H), 7.42–7.27 (m, 6H), 6.75 (s, 1H),
2.39 ppm (s, 2H); 13C NMR (CDCl3): d=198.4, 143.5, 134.4, 130.5,
128.9, 128.2, 128.1, 127.6, 127.5, 127.1, 126.9, 126.6, 27.5 ppm.
hydropyrimido[5,4-f][1,2,4]triazolo[3,4-b][1,3,4]
thiadiazepine
(25): To a suspension of compound 24 (50 mg, 0.1 mmol) in abso-
lute ethanol (2 mL) was added sodium borohydride (3.8 mg,
0.1 mmol) in one portion with stirring. The yellow solution formed
was kept at room temperature for 2 h. The solvent was removed
under reduced pressure, and the residue was washed with water
and filtered. The resulting white solid was collected to give com-
2-(3,5-Bis(trifluoromethyl)phenyl)-5-((4,6-dichloropyrimidin-2-
yl)thio)-1,3,4-oxadi-azole (17): Synthesis of 17 followed the same
1
pound 25 (41 mg, 82%). H NMR (CDCl3): d=8.72 (s, 2H), 8.03 (s,
1
procedure as for 7a in 62% yield as a white solid. H NMR (CDCl3):
1H), 6.27 (t, J=6.2 Hz, 1H), 4.52 (d, J=6.2 Hz, 1H), 2.51 ppm (s,
3H); HRMS (ESI) calcd for C16H10ClF6N6S2: 498.9996, found:
498.9991 [M+H]+.
d=8.57 (s, 2H), 8.12 (s, 1H), 7.66 ppm (s, 1H); 13C NMR (CDCl3): d=
167.6, 165.6, 162.7, 160.5, 157.4, 133.4, 127.2, 125.9, 124.9, 123.9,
121.2, 117.0 ppm; HRMS-ESI (+): calcd for C14H5Cl2F6N4OS:
460.9445, found: 460.9465 [M+H]+.
3-(3,5-Bis(trifluoromethyl)phenyl)-9-(methylthio)-5,6,7,8-tetrahy-
dropyrimido[5,4-f][1,2,4]triazolo[3,4-b][1,3,4] thiadiazepine (26):
To a suspension of compound 24 (50 mg, 0.1 mmol) in absolute
ethanol (2 mL) sodium borohydride (38 mg, 1 mmol) was added in
portions with stirring. The yellow solution formed was kept at
room temperature for 2 h. The solvent was removed under re-
duced pressure, and the residue was washed with water and then
filtered. The resulting white solid was collected to give compound
2-((3-(3,5-Bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-5-yl)oxy)-4-
((5-(3,5-bis(trifluoro methyl)phenyl)-4H-1,2,4-triazol-3-yl)oxy)-6-
chloropyrimidine (18): Synthesis of 18 followed the same proce-
1
dure as for 7a in 65% yield as a white solid. H NMR (CDCl3): d=
8.53 (s, 4H), 8.12 ppm (s, 2H); 13C NMR (CDCl3): d=167.3, 165.5,
160.3, 157.8, 133.3, 133.0, 127.2, 125.9, 124.9, 123.9, 121.2,
14.1 ppm.
1
26 (20 mg, 40%). H NMR (CDCl3/CD3OD 1:1): d=8.72 (s, 2H), 8.02
(s, 1H), 3.69 (d, J=8.2 Hz, 4H), 2.51 ppm (s, 3H); HRMS (ESI) calcd
for C16H13F6N6S2: 467.0542, found: 467.0551 [M+H]+.
4-(4-Bromophenyl)-2-((4,6-dichloropyrimidin-2-yl)thio)-6-oxo-1,6-
dihydropyrimidine-5-carbonitrile (20a): Synthesis of 20a followed
the same procedure as for 7a in 45% yield as a white solid.
1H NMR (CDCl3): d=7.82–7.80 (d, J=8.4 Hz, 2H), 7.66 (d, J=8.4 Hz,
2H), 7.56 ppm (s, 1H); HRMS-ESI (+): calcd for C15H7BrCl2N5OS:
453.8855, found: 453.8932 [M+H]+.
Biology
Bacterial strains and culture conditions: E. coli NR698 (MC4100
imp4213),[19] an outer membrane leaky mutant strain, was kindly
provided by Thomas J. Silhavy (Princeton University). S. aureus
ATCC strains 6538 and 35556 were from the American Type Culture
Collection. S. aureus strains Mu50, Mu3, and N315 were kindly pro-
vided by C.-D. Lu, and Newman strain from Z. Eichenbaum (Geor-
gia State University). Five efflux-pump-related S. aureus strains
2-((4,6-Dichloropyrimidin-2-yl)thio)-4-(4-ethylphenyl)-6-oxo-1,6-
dihydropyrimidine-5-carbonitrile (20b): Synthesis of 20b fol-
lowed the same procedure as for 7a in 52% yield as a white solid.
1H NMR (CDCl3): d=7.93 (s, 1H), 7.72–7.70 (d, J=7.6 Hz, 2H), 7.43–
7.41 (d, J=7.6 Hz, 2H), 7.38 (s, 1H), 2.80–2.74 (m, 2H), 1.31–
1.24 ppm (m, 3H).
8325-4, K1758 (NorAÀ), K2361 (NorA+ +), K2908 (MepAÀ), and
[1d,20]
K2068 (MepA+ +
)
were kindly provided by G. W. Kaatz (Wayne
4-Amino-5-(3,5-bis(trifluoromethyl)phenyl)-4H-1,2,4-triazole-3-
thiol (22): To a mixture of 21, 3,5-bis(trifluoromethyl)benzhydrazide
(2.7 g, 10 mmol) and carbon disulfide (1.14 g, 15 mmol) in 20 mL
absolute ethanol was added KOH (0.84 g, 15 mmol) pellets, and
the reaction was held at reflux for 6 h. The solvent was evaporated
under vacuum to afford potassium dithiocarbazate as a white
solid, which was treated with hydrazine monohydrate (1.6 mL,
25 mmol) in water and then heated at reflux for 4 h. The color of
the reaction mixture changed to green with the evolution of hy-
drogen sulfide gas (lead acetate paper test and odor). Then the re-
action mixture was cooled to room temperature, diluted with
water (100 mL), and acidified with concentrated HCl. The resulting
precipitate was filtered, washed thoroughly with cold water, and
recrystallized from ethanol to give compound 22 as a white solid
State University School of Medicine). Bacillus subtilis 168 and Bacil-
lus anthracis Sterne were lab stocks. All strains were grown on
Luria–Bertani (LB) broth or agar plates at 378C.
Chemicals: DMSO was purchased from Sigma. The initial 38 com-
pounds for screening, including hit compound 1, were purchased
from Maybridge.
Protein purification: EcSecA, SpSecA, and BsSecA were purified as
described.[21] EcSecAN68, a truncated mutant of EcSecA containing
the N-terminal catalytic domain, was purified as previously descri-
bed.[16b,21b] The SasecA1 gene was amplified from S. aureus
ATCC35556 and cloned into pET-21d with a His6 tag at the C termi-
nus. The BasecA1 gene was amplified from B. anthracis Sterne, and
cloned into pET-20b with a C-terminal His6 tag. SaSecA1 and
BaSecA1 were purified with a HisTrap affinity column and a Super-
dex-200 column (GE healthcare).
1
(2.62 g, 82%). H NMR (DMSO): d=14.2 (s, 1H), 8.69 (s, 2H), 8.31 (d,
J=7.6 Hz, 1H), 5.86 ppm (s, 3H); HRMS-ESI calcd for C10H7F6N4S:
329.0290, found: 329.0294 [M+H]+.
3-(3,5-Bis(trifluoromethyl)phenyl)-7-chloro-9-(methylthio)pyrimi-
do[5,4-f][1,2,4]triazolo[3,4-b][1,3,4] thiadiazepine (24): A mixture
of compound 22 (328 mg, 1 mmol) and compound 23, 4,6-di-
Liposome and membrane preparations: The preparation of lipo-
somes was performed as described previously.[8] Briefly, E. coli total
lipids (Avanti) were dried and resuspended in 150 mm KCl solution,
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ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
and sonicated in an ice-water bath until the solution became clear,
usually 3–5 min, which resulted in an average liposome diameter
of ~130 nm. The liposome solutions were aliquoted, and stored at
À808C before use. The liposomes were frozen and thawed once
for the experiments. The preparation of SecA-depleted native
membranes from BA13 is as previously described.[18a,22] The BA13
cells were grown at 428C to deplete the SecA, and the cells were
collected and lysed by French press as described. E. coli 773 is an
ompA-deleted strain in lab stock; the membrane vesicles were pre-
pared from sucrose gradients as described.[23]
buffer containing EDTA-free cocktail protease inhibitors. The cells
were broken by French press at 69 MPa. Unbroken cells were re-
moved by centrifugation at 5000 rpm (3000g). Whole-cell lysates
were treated with PureProteome Streptavidin Magnetic Beads (Mil-
lipore) at 48C for 1 h to remove nonspecific binding. The superna-
tant was mixed beads with or without 400 mm SCA-256 at 48C for
1 h. The beads were washed with TBST buffer three times and
were mixed with SDS sample buffer, and boiled for 15 min. Cross-
reacting EcSecA antibody (lab stock) was used to detect SaSecA1
by western blot.
ATPase assays for EcSecAN68 and EcSecA: ATPase activities were de-
termined by malachite green colorimetric assay as described pre-
viously.[15,16b] Translocation ATPase was assayed in the presence of
SecA-depleted membranes and proOmpA precursors. IC50 is the
concentration of the compound that inhibits 50% of ATPase activi-
ties.
Acknowledgments
Financial support by the US National Institutes of Health
(AI104168) and Fellowships from the Molecular Basis of Diseases
Program at Georgia State University (J.S., A.S.C., Y.H.H., and W.C.)
is gratefully acknowledged.
In vitro translocation activity of EcSecA: In vitro translocation activity
of EcSecA was determined by using OmpA-depleted 773 mem-
branes and SecA–liposome as described previously.[8] Briefly, recon-
stituted liposomes, SecA, and proOmpA were added into the trans-
location buffer[8] separately before reaction. Liposomes at 12 mg or
4.5 mg of OmpA-depleted 773 membranes with various amounts of
SecA were used in the 0.1 mL reaction mixtures. The reactions
were carried out at 378C for 45 min, and the translocation mixtures
were processed by immunoblots as previously described.[8] IC50 is
the concentration of the compound that inhibits 50% of in vitro
translocation activity.
Keywords: antimicrobials · protein secretion · pyrimidines ·
SecA inhibitors · triazoles
[1] a) H. W. Boucher, G. H. Talbot, J. S. Bradley, J. E. Edwards, D. Gilbert, L. B.
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Ion channel activity of SecA–liposomes in oocytes: Liposomes were
prepared as described previously.[8,9,15,16b,24] Oocytes were obtained
from live frog Xenopus laevis (Xenopus Express, Inc.) and injected
with sample mixtures as described previously.[8,18b] Briefly, 50 nL
sample mixtures containing 120 ng liposomes, 120 ng SecA, 14 ng
proOmpA, 2 mm ATP, 1 mm Mg(OAC)2, and various concentrations
of inhibitors were injected into oocytes (average volume: 500 nL)
using a Nanoject II injector. The voltage clamp adapted from an
electrophysiological method was used to measure the opening of
protein conducting channels as described previously.[8,18b] After in-
jection, the oocytes were incubated at 238C for 3 h, then the ion
current was recorded continuously for 1 min. The inward and out-
ward currents were recorded to measure the net currents for chan-
nel opening. IC50 is the concentration of the compound that inhib-
its 50% ion channel activities of SaSecA1.
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Carmelic, M. E. Falagasd, C. G. Giskef, S. Harbarthg, J. F. Hindlerh, G.
Kahlmeteri, B. Olsson-Liljequistj, D. L. Patersonk, L. B. Ricel, J. Stellingm,
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Bacteriostatic effect: Bacteriostatic effects were tested according to
the guidelines of the Clinical and Laboratory Standards Institute.[25]
This assay was performed in a 96-well microtiter plate in triplicate
in three separate experiments, as described previously, at 378C
with shaking at 250 rpm for 24 h. MIC is the lowest concentration
of inhibitor at which cells were unable to grow.
Bactericidal effect: Bactericidal effect was determined as described
previously.[16b] Cells in log-phase growth (OD600 ꢁ0.5) were mixed
with various concentrations of inhibitors, and then incubated in an
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Received: September 29, 2015
Published online on November 26, 2015
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