The first low μM SecA inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.12.074

SecA ATPase is a critical member of the Sec family, which is important in the translocation of membrane and secreted polypeptides/proteins in bacteria. Small molecule inhibitors can be very useful research tools as well as leads for future antimicrobial agent development. Based on previous virtual screening work, we optimized the structures of two hit compounds and obtained SecA ATPase inhibitors with IC₅₀ in the single digit micromolar range. These represent the first low micromolar synthetic inhibitors of bacterial SecA and will be very useful for mechanistic studies.

Full text of DOI:10.1016/j.bmc.2009.12.074

Bioorganic & Medicinal Chemistry 18 (2010) 1617–1625
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The first low lM SecA inhibitors
Weixuan Chen a, Ying-ju Huang b, Sushma Reddy Gundala b, Hsiuchin Yang b, Minyong Li a,
a,
Phang C. Tai b, , Binghe Wang
*
*
a Department of Chemistry, Georgia State University, Atlanta, GA 30302-4098, USA
b Department of Biology, Georgia State University, Atlanta, GA 30302-4098, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
SecA ATPase is a critical member of the Sec family, which is important in the translocation of membrane
and secreted polypeptides/proteins in bacteria. Small molecule inhibitors can be very useful research
tools as well as leads for future antimicrobial agent development. Based on previous virtual screening
work, we optimized the structures of two hit compounds and obtained SecA ATPase inhibitors with
IC50 in the single digit micromolar range. These represent the first low micromolar synthetic inhibitors
of bacterial SecA and will be very useful for mechanistic studies.
Received 7 October 2009
Revised 21 December 2009
Accepted 31 December 2009
Available online 6 January 2010
Keywords:
SecA inhibitor
2-Thiouracil
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
antimicrobial agents, especially because SecA has no human coun-
terpart. We have previously reported effort in using virtual screen-
With the rapid emergence of drug resistant bacteria, there is an
urgent need for the development of new antimicrobial agents,
especially those with a unique mechanism of action. With this ulti-
mate goal in mind, we are interested in the development of inhib-
itors of bacterial protein translocation. Several protein transport
mechanisms exist in bacteria.1 Among them, the Sec machinery
(or translocase) provides a major pathway of protein translocation
from the cytosol across or into the cytoplasmic membrane. The Sec
machinery has seven proteins including SecA, SecD, SecE, SecF,
SecG, SecY, and YajC. Assembly and complex formation are re-
quired to yield the functional translocase. Among the Sec proteins,
SecA is found both in the cytoplasm and bound to the inner mem-
brane. When SecA is bound to the SecYEG complex, acidic phos-
ing against the Escherichia coli SecA crystal structure9 to search for
possible structural features suitable for SecA inhibitor develop-
ment.10 In this paper, we describe our effort in optimizing the
structural features of the initial hits for the development of bacte-
rial SecA inhibitors. Several low
lM inhibitors have been found.
Considering the fact that currently inorganic azide, which is a SecA
inhibitor with an IC50 value of about 3 mM, has cross reactivities
against a number of enzymes,11,12 and is the primary research tool
for probing bacterial protein translocation, the newly discovered
SecA inhibitors will be very important.
2. Results and discussions
2.1. Chemistry
pholipids and
a precursor protein such as proOmpA (the
precursor of outer membrane protein A), it becomes fully active
as an ATPase and a protein translocase.2,3 Recently, several seminal
papers described in intricate details as to how the SecA machinery
functions in transporting proteins.4–6
It has been said that in any given organism, membrane and se-
creted polypeptides/proteins comprise more than 30% of the prote-
ome; and no less than 10% of proteins cross a membrane before
arriving at their final locations of function.7,8 Such actions are often
mediated by protein translocases. Therefore SecA is essential for
bacterial survival. We envision that inhibitors of SecA can be very
useful tools for studying bacterial protein transport and potential
In our earlier virtual screening efforts, two hits, 1 (SEW-05929)
and 2 (HTS-12302), were shown to have modest SecA inhibitory
activities (IC50 values of about 100 l
M).10,13 Since there were no
other known SecA inhibitors except one natural product, for which
the true inhibition mechanism was not known,14 our effort to
search for potent SecA inhibitors started with the optimization of
these two modest inhibitors (Fig. 1).
Our optimization effort first started with the isoxazole carbox-
amide series (1) with the focus being on optimizing the aryl group
attached to the amide. In this series, 14 analogs were synthesized.
The synthesis started with conversion of halogenated benzalde-
hyde 3 to the corresponding oxime 4 (Scheme 1). Isoxazole acid
6 was prepared by reacting 5 with ethyl acetoacetate followed by
hydrolysis.15 Subsequent coupling/amidation reactions using EDCI
* Corresponding authors. Tel.: +1 404 413 5303; fax: +1 404 413 5301 (P.C.T.);
tel.: +1 404 413 5544; fax: +1 404 413 5543 (B.W.).
E-mail addresses: biopct@langate.gsu.edu (P.C. Tai), wang@gsu.edu (B. Wang).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.074

1618
W. Chen et al. / Bioorg. Med. Chem. 18 (2010) 1617–1625
S
N
For this series, we were interested in further simplifying the struc-
S
N
S
S
ture to understand the core structural need. Therefore, ‘monomer’
series 17d,e,g–i was prepared by benzylation of compounds
14d,e,g–i with benzyl chloride, and the difference in activities be-
tween the ‘dimer’ and ‘monomer’ series was also studied.
N
S
S
Cl
O
2
NH
N
F
O
2.2. Biological evaluation
S
O
CN
1
N
R'
NH
O
HN
S
2.2.1. In vitro study
R'
N
The synthesized compounds were first evaluated using EcN68
SecA, which is a truncated version without the C-terminal regula-
tory/inhibitory domain, by following procedures published ear-
lier.10 Briefly, ATPase activities were determined by the release of
phosphate (Pi), which can be detected spectrophotometrically
using malachite green.14 For compounds 7a–n, none of them
showed improved activities over the original hit (1) or significant
NC
R'
Cl
N
S
Cl
NH
N
O
NH
O
R
O
NC
inhibition at 100 lM (Fig. 2). Such results coupled with the weak
activities of the original hit compound led to the decision of not
Figure 1. Two hit compounds and their derivatives.
pursuing this class of compounds any further.
For analogs of 2, compounds 15a–g did not show significant
improvement over the initial hit (data not shown). However, the
substituted thiouracils (16) showed very significant activities
and DMAP gave the final isoxazole carboxamide derivatives 7a–7n.
In this series, there were amides of aniline compounds 7a–g, pri-
mary alkylamines 7h,i, secondary alkyamines 7j–l, and benzylam-
ines 7m,n.
when screened at 100 (data not shown) and 30
compounds that showed potent inhibition at 30
screened at 5 M (Fig. 4). Within the symmetrical compound series
lM (Fig. 3). Those
lM were further
In optimizing the second series (2, Fig. 1), we first started by
testing different aryl structures flanking the central ring. In our ini-
tial effort, 6-chloro-2-mercaptobenzothiazole and 2-mercapto-
benzoxazole derivatives were prepared by reacting potassium
ethylxanthate 8 with 2,4-dichloroaniline 9 or substituted 2-amino-
phenol 10 (Scheme 2). Further, 5-cyano-6-aryl-2-thiouracils were
prepared ‘by condensation of an aldehyde with ethyl cyanoacetate
and thiourea in the presence of piperidine’.16 The symmetrical
compounds 15a–g or 16a–i were obtained by reacting 2 equiv of
compounds 11a–g or 14a–i with p-xylylene dibromide in acetoni-
trile in the presence of K2CO3 (Scheme 3). One successful series of
l
16a–i, there were two substitution patterns: one with a phenyl
ring substituted at the 4-position and the other with a phenyl ring
substituted at the 3-position. The results showed that the 4-substi-
tuted analogs were more potent than the 3-substituted class,
which was in turn slightly more potent than the ones without phe-
nyl substituent. For example the activities of the 4-methyl substi-
tuted (16c, Fig. 3) was higher than the 3-methyl analog (16b,
Fig. 3), which was in turn higher than the unsubstituted one
(16a, Fig. 3). With the initial indication that derivatives with a phe-
nyl ring bearing a 4-substituent were more active, the subsequent
effort was focused on optimizing this series of compounds. One
approach adopted was to use relatively bulky alkyl groups at the
analogs was the 2,20-(
a,
a0-xylene)bis(sulfanediyl)bis-(6-aryl-5-cy-
ano-4-oxopyrimidine) 16a–i (see below for biological results).
O
O
Cl
Cl
Cl
N
N
Cl
Cl
a
e
b
c, d
O
N
N
R
OH
OH
COOH
O
Cl
Cl
Cl
Cl
Cl
6
Cl
7a-7n
3
4
5
Compound
Compound
Compound
R
R
R
HN
HN
HN
7a
7b
7c
HN
HN
F
HN
7e
7d
7g
7f
Cl
Br
F
HN
HN
HN
7h
7i
7l
Cl
N
7j
7k
O
N
S
N
HN
HN
7m
7n
Br
Br
Scheme 1. Synthesis of isoxazole carboxamides 7a–n. Reagents and conditions: (a) HONH2ꢀHCl, NaOH, EtOH, H2O, reflux; (b) NCS, DMF; (c) ethyl acetoacetate, MeONa, THF;
(d) NaOH, EtOH, H2O; (e) EDCI, HOBt, DMAP, DMF.

W. Chen et al. / Bioorg. Med. Chem. 18 (2010) 1617–1625
1619
NH2
Cl
N
S
a
a
SH
b R'' = H
c R'' = 4-methyl
d R'' = 5-methyl
e R'' = 3-nitro
f R'' = 5-nitro
g R'' = 4-chloro
Cl
Cl
9
11 a
Et-O-CS2HK
+
or
8
NH2
OH
N
O
R''
R''
SH
10
11 b - g
a R' = H
b R' = 3-methyl
c R' = 4-methyl
d R' = 4-ethyl
e R' = 4-i-propyl
S
O
b
S
OEt
HN
NH
NC
H
H2N
NH2
f
R' = 4-methoxyl
O
O
g R' = 4-bromo
h R' = 4-phenyl
i R' = 1-naphthalenyl
CN
14 a - i
R'
12
13
R'
Scheme 2. Synthesis of compounds 11a–g and 14a–i. Reagents: (a) EtOH, reflux; (b) piperidine, EtOH, reflux.
Cl
S
S
11 a - g
N
O
N
N
S
a
Br
or
S
S
Br
15 a
Cl
14 a - i
b R'' = H
c R'' = 4-methyl
d R'' = 5-methyl
e R'' = 3-nitro
R''
R'
S
N
O
a R' = H
b R' = 3-methyl
c R' = 4-methyl
d R' = 4-ethyl
e R' = 4-i-propyl
R' = 4-methoxyl
g R' = 4-bromo
h R' = 4-phenyl
R''
15 b - g
S
O
HN
S
CN
f
R'' = 5-nitro
N
g R'' = 4-chloro
f
NH
O
R'
N
i R' = 1-naphthalenyl
NC
a
16 a - i
S
S
HN
NH
Cl
N
NH
O
O
CN
14 d,e,g-i
CN
R'
R'
17 d,e,g-i
Scheme 3. Synthesis of compounds 15a–g, 16a–i and 17d,e,g–i. Reagents: (a) K2CO3, CH3CN, reflux.
125
100
75
50
25
0
125
100
75
50
25
0
Figure 2. Inhibitory effect of compounds 1 and 7a–n at 100 lM against EcN68 Sec
A.
Figure 3. Inhibitory effect of compounds 16a–i at 30 lM against EcN68 Sec A.
4-position. It turned out that these compounds were more potent
than the corresponding 4-methyl substituted compounds. Among
these compounds, those with an electron donating (e.g., methoxy)
substituent seemed to be less active than the unsubstituted ones
also tested (Fig. 5). First of all, both thiouracil-based ‘dimer’ and
‘monomer’ compounds showed more potent inhibition than the
benzothiazole or benzoxazole compounds 15a–g. However, the ‘di-
mer’ series 16d,e,g–i were more potent than the ‘monomer’ series
17d,e,g–i, respectively. This higher potency for the ‘‘dimer” series
seems to come from better fitting of the binding pocket of these
compounds (see below). In the ‘monomer’ series, it was observed
that a large sized R0 group seemed to confer high potency (e.g.,
17h > 17g ꢁ 17e > 17d). However, when the substituent phenyl
(e.g., 16f < 16a, Fig. 3). At 5 lM, analogs with a halogen or aryl
group substitution at the 4-position were more potent than the
analogues with an alkyl substitution (e.g., 16g,h,i > 16c,d,e
Fig. 4). For the examination of the difference between the ‘dimers’
and ‘monomers’, S-benzyl-2-thiouracils analogues 17d,e,g–i were

1620
W. Chen et al. / Bioorg. Med. Chem. 18 (2010) 1617–1625
125
100
75
50
25
0
150
16g
16h
17h
100
50
Figure 4. Inhibitory effect of compounds 16c–e,g–i at 5 lM against EcN68 Sec A.
125
100
75
50
25
0
0
1
10
100
1000
concentration (µM)
Figure 7. The inhibitory curves of 16g,h and 17h against EcSecA.
16h against NR698
17h against NR698
17h against MC4100
125
Figure 5. Inhibitory effect of compounds 17d,e,g–i at 30 lM against EcN68 Sec A.
100
75
50
25
0
ring was replaced by a larger 1-naphthyl group, the activity
seemed to decrease slightly.
We determined the IC50 values of compound 16g and 16h since
they showed the most potent activities of all the compounds
screened at 5
inhibition (IC50: 2
the hit compound 2 (IC50: 100
Inhibition tests using whole EcSecA gave similar results (16g
IC50: 20 M, 16h IC50: 50 M and 17h IC50: 60 M, Fig. 7), which
l
M. The result showed they had low micro molar
M, Fig. 6), which is 50-fold more potent than
M).10
l
l
1
10
100
l
l
l
concentration (µM)
suggested that the EcN68 inhibition assay was more sensitive than
the whole SecA inhibition assay. This is understandable since EcS-
ecA contains a regulatory domain, which is essentially an inhibitor.
Figure 8. The inhibitory curves of 16g,h and 17h against bacterial growth.
2.2.2. In vivo study
might be a key factor and for in vivo applications future studies
should focus on low molecular weight compounds such as 17h
for structural optimization.
The biological activities of ‘dimer’ and ‘monomer’ compounds
16h and 17h were assessed against leaky mutant NR698 and wild
type E. coli strain MC4100 by determining the minimum inhibition
concentration (MIC) (Fig. 8). ‘Monomer’ compound 17h exhibited
the most potent inhibition effects against NR698, whereas ‘dimer’
compounds 16h did not exhibit significantly antimicrobial activi-
ties. However, neither 17h nor 16h exhibited inhibition effects
against wild type E. coli strain MC4100. Such results suggested that
the permeability of 16h against NR698 and 17h against MC4100
2.3. Computational modeling
In order to achieve a detailed understanding the binding mode
between SecA and our compounds, in silico modeling was con-
ducted by using molecular simulation.17–20 Herein, the parent
compound, HTS-12302 and the most active compound, 16g, were
docked into the ATP-site of SecA using DOCK 5.4. The docked com-
plexes were then optimized by molecular mechanics and molecu-
lar dynamics simulation implemented in AMBER 8. Finally, the
possible ligand–protein interactions were examined by following
similar procedures we used in previous studies.10 After molecular
simulation, compound HTS-12302 seems to bind SecA through
interactions with Thr 104 by forming hydrogen bond and with
Met 81, Phe 84, Gln 87, Gly 105, Glu 106, Gly 107, Lys 108, Thr
109, Leu 110, Gly 392 and Arg 509 through hydrophobic interac-
tions (Fig. 9). Compound 16g has a similar binding conformation
and orientation, in which it seems to engage in more hydrogen
bond interactions with Phe 84, Gln 87, Lys 108 and Glu 210. More-
over, compound 16g still bears hydrophobic interactions with Met
81, Thr 104, Gly 105, Glu 106, Gly 107, Leu 110 and Arg 509. Upon
analysis of the structural features of these two compounds, it
seems that the inclusion of the thiouracil moiety may contribute
to the inhibitory activity because of more hydrophobic interaction
and hydrogen bonds when compared with lead compound
125
100
16g
16h
75
50
25
0
0.1
1
10
100
concentration (µM)
Figure 6. The inhibitory curves of the two most potent compounds, 16g and 16h,
against EcN68 Sec A.

W. Chen et al. / Bioorg. Med. Chem. 18 (2010) 1617–1625
1621
further structural optimization in search of even more potent
inhibitors as potential antimicrobial agents.
3. Experimental
3.1. Chemistry
3.1.1. General chemical methods
All reagents and solvents were reagent grade or were purified
by standard methods before use. Column chromatography was car-
ried out on flash silica gel (Sorbent 230–400 mesh). TLC analysis
was conducted on silica gel plates (Sorbent Silica G UV254). NMR
spectra were recorded at 1H (400 MHz) and 13C (100 MHz) with a
Bruker instrument. Chemical shifts (d values) and coupling con-
stants (J values) are given in ppm and hertz, respectively, using
TMS (1H NMR) and solvents (13C NMR) as internal standards.
3.1.2. General procedure for the preparation of isoxazole
carboxamide derivatives (7a–7n)
Under N2 atmosphere, a solution of an isoxazole carboxylic acid
(6, 0.1 mmol), amine (0.12 mmol), EDCI (23 mg, 0.12 mmol), DMAP
(14.7 mg, 0.12 mmol) and HOBt (27 mg, 0.2 mmol) in DMF (2.5 mL)
was stirred at room temperature overnight. Then most of the sol-
vent was removed under reduced pressure. To the residue was
added 10 mL H2O and 10 mL EtOAc. Then the aqueous solution
was extract by EtOAc (20 mL ꢂ 2). The organic layer was subse-
quently washed with brine (20 mL). The crude compound was
purified by flash chromatography on silica gel using hexane and
EtOAc (9:1) as the mobile phase to give 7a–7n.
3.1.2.1. 3-(2,6-Dichlorophenyl)-5-methyl-N-m-tolylisoxazole-4-
carboxamide (7a). Yield 76%; 1H NMR (CDCl3) d 1.70 (s, 3H), 2.86
(s, 3H), 6.74 (br s, 1H), 7.00–7.07 (m, 2H), 7.18 (td, 1H, J = 1.6 Hz,
7.2 Hz), 7.44 (dd, 1H, J = 6.4 Hz, 9.6 Hz), 7.50 (m, 2H), 7.90 (d, 1H,
J = 8.0 Hz); 13C NMR (CDCl3) d 13.7, 16.9, 112.0, 123.0, 125.4,
127.0, 127.3, 128.3, 129.1, 130.6, 132.7, 135.3, 136.6, 155.7,
159.0, 176.4. HRMS-ESI (+): Calcd for C18H15N2O2Cl2: 361.0511.
Found: 361.0527 [M+H]+.
3.1.2.2. N-(3-Bromobenzyl)-3-(2,6-dichlorophenyl)isoxazole-4-
carboxamide (7b). Yield 71%; 1H NMR (CDCl3) d 2.27 (s, 3H), 2.84
(s, 3H), 6.85 (m, 3H), 7.11 (m, 2H), 7.48 (dd, 1H, J = 6.4 Hz, 9.6 Hz),
7.54 (m, 2H); 13C NMR (CDCl3) d 13.5, 21.7, 112.1, 117.1, 120.8,
125.8, 127.3, 129.0, 129.0, 132.7, 136.4, 137.2, 139.3, 155.9,
158.9, 175.7. HRMS-ESI (+): Calcd for C18H15N2O2Cl2: 361.0511.
Found: 361.0516 [M+H]+.
Figure 9. (A) The proposed docking conformation of HTS-12302 (white sticks) and
compound 16g (green sticks) around SecA ATP-site; (B) the proposed schematic
interactions of HTS-12302 with SecA; and (C) the proposed schematic interactions
of compound 16g with SecA.
3.1.2.3. (3-(2,6-Dichlorophenyl)-5-methylisoxazol-4-yl)(mor-
pholino)methanone (7c). Yield 81%; 1H NMR (CDCl3) d 1.63 (s,
3H), 2.18 (s, 3H), 2.80 (s, 3H), 6.62 (br s, 1H), 6.82 (s, 1H), 6.92
(d, 1H, J = 8.4 Hz), 7.37 (dd, 1H, J = 6.4 Hz, 9.6 Hz), 7.44 (m, 2H),
7.66 (d, 1H, J = 8.4 Hz); 13C NMR (CDCl3) d 13.7, 16.9, 21.0, 112.0,
123.2, 127.4, 127.5, 128.6, 129.0, 131.2, 132.7, 135.3, 136.6,
155.7, 159.0, 176.2. HRMS-ESI (+): Calcd for C19H17N2O2Cl2:
375.0667. Found: 375.0679 [M+H]+.
HTS-12302. Such structural insights will play a very critical role in
future design of potent SecA inhibitors and in further structural
optimizations.
2.4. Conclusion
Through optimization of two hit compounds 1 (SEW-05929)
and 2 (HTS-12302) identified from virtual screening, we have
found a series of thiouracil derivatives that are much more potent
than the primary hits. The two most potent compounds, 16g and
16h, are 50-fold more active than the hit compounds. Results of
antimicrobial tests suggest that future work should focus on low
molecular weight analogs of 17h for in vivo applications. These
compounds are the first in its class and should be very useful as re-
search tools in studying bacterial protein transport. The new inhib-
itory structural features identified should also be very useful for
3.1.2.4. (3-(2,6-Dichlorophenyl)-5-methylisoxazol-4-yl)(piperi-
din-1-yl)methanone (7d). Yield 67%; 1H NMR (CDCl3) d 2.24 (s,
3H), 2.78 (s, 3H), 6.70 (dd, 1H, J = 2.4 Hz, 8.4 Hz), 6.75 (br s, 1H),
7.12 (d, 1H, J = 2.4 Hz), 7.29 (d, 1H, 8.8 Hz), 7.43 (dd, 1H,
J = 6.4 Hz, 9.6 Hz), 7.49 (m, 2H), 7.62 (m, 5H); 13C NMR (CDCl3) d
13.6, 23.3, 111.9, 118.8, 120.1, 122.2, 127.2, 129.0, 132.8, 132.8,
136.3, 136.5, 139.0, 155.8, 158.8, 175.9. HRMS-ESI (+): Calcd for
C18H14N2O2Cl2Br: 438.9616. Found: 438.9633 [M+H]+.

1622
W. Chen et al. / Bioorg. Med. Chem. 18 (2010) 1617–1625
3.1.2.5. 3-(2,6-Dichlorophenyl)-N-(2,4-dimethylphenyl)-5-methyl-
isoxazole-4-carboxamide (7e). Yield 55%; 1H NMR (CDCl3) d 2.87 (s,
3H), 6.92–6.99 (m, 2H), 7.08 (t, 1H, J = 7.6 Hz), 7.20 (br s, 1H), 7.47 (dd,
1H, J = 6.4 Hz, 9.6 Hz), 7.52 (m, 2H), 8.33 (td, 1H, J = 1.6 Hz, 8.0 Hz);
13C NMR (CDCl3) d 13.7, 111.8, 114.7, 114.9, 121.6, 124.7, 124.8,
124.8, 124.8, 126.3, 129.1, 132.8, 136.3, 153.4, 155.9, 158.8, 176.4.
HRMS-ESI (+): Calcd for C17H12N2O2Cl2F: 365.0260. Found: 365.0269
[M+H]+.
HRMS-ESI (+): Calcd for C15H15N2O2SCl2: 357.0231. Found:
357.0237 [M+H]+.
3.1.2.13. N-(2-Bromobenzyl)-3-(2,6-dichlorophenyl)-5-methy-
lisoxazole-4-carboxamide (7m). Yield 73%; 1H NMR (CDCl3) d
2.73 (s, 3H), 4.37 (d, 2H, J = 6.0 Hz), 5.62 (br s, 1H), 7.04 (m, 1H),
7.16 (m, 2H), 7.26–7.33 (m, 3H), 7.38 (d, 1H, J = 8.0 Hz); 13C NMR
(CDCl3) d 13.4, 43.9, 111.6, 123.9, 127.3, 127.9, 128.8, 129.5,
130.6, 132.3, 132.8, 136.2, 136.9, 156.1, 160.6, 175.2. HRMS-ESI
(+): Calcd for C18H14N2O2Cl2Br: 438.9616. Found: 438.9627
[M+H]+.
3.1.2.6. N-(3-Chlorophenyl)-3-(2,6-dichlorophenyl)-5-methylis-
oxazole-4-carboxamide (7f). Yield 42%; 1H NMR (CDCl3) d 2.79
(s, 3H), 6.81 (br s, 1H), 6.84 (m, 1H), 6.98 (m, 1H), 7.09 (t, 1H,
J = 8.0 Hz), 7.30 (t, 1H, J = 1.6 Hz), 7.45(dd, 1H, J = 6.4 Hz, 9.6 Hz),
7.50 (m, 2H); 13C NMR (CDCl3) d 90.2, 113.8, 128.2, 128.6, 132.0,
158.2, 160.2, 176.0. HRMS-ESI (+): Calcd for C17H12N2O2Cl3:
380.9964. Found: 380.9962 [M+H]+.
3.1.2.14. 3-(2,6-Dichlorophenyl)-5-methyl-N-(4-methylcyclo-
hexyl)isoxazole-4-carboxamide (mixture of cis and trans)
(7n). Yield 93%; 1H NMR (CDCl3) d 2.74 (s, 3H), 4.28 (d, 2H,
J = 5.6 Hz), 5.27 (br s, 1H), 6.91 (d, 1H, J = 7.2 Hz), 7.04 (m, 2H),
7.29 (d, 2H, J = 7.6 Hz), 7.34 (d, 2H, J = 7.6 Hz); 13C NMR (CDCl3) d
13.4, 42.9, 111.5, 122.9, 126.2, 127.3, 128.8, 130.2, 130.4, 132.5,
136.0, 139.8, 156.1, 160.7, 175.2. HRMS-ESI (+): Calcd for
C18H14N2O2Cl2Br: 438.9616. Found: 438.9635 [M+H]+.
3.1.2.7. N-Cyclohexyl-3-(2,6-dichlorophenyl)-5-methylisoxazole-
4-carboxamide (7g). Yield 59%; 1H NMR (CDCl3) d 2.83 (s, 3H),
6.83 (br s, 1H), 6.87 (m, 1H), 6.98 (t, 1H, J = 8.8 Hz), 7.42 (dd, 1H,
J = 2.8 Hz, 6.8 Hz), 7.50 (dd, 1H, J = 6.4 Hz, 9.6 Hz), 7.55 (m, 2H);
13C NMR (CDCl3) d 13.6, 111.7, 116.8, 117.0, 119.6, 119.7, 121.4,
121.6, 122.4, 127.1, 129.1, 132.9, 133.8, 136.3, 154.0, 155.7,
156.4, 158.9, 176.1. HRMS-ESI (+): Calcd for C17H11N2O2FCl3:
398.9870. Found: 398.9885 [M+H]+.
3.1.3. General procedures for the preparation of and 2-
mercaptobenzoxazole (11b–11g)
To a solution of a substituted 2-aminophenol (3 mmol) was
added potassium ethylxanthate (484 mg, 3 mmol) in absolute eth-
anol (10 mL). The resulting mixture was heated under reflux over-
night and then cooled to room temperature. The precipitate was
dissolved in H2O (10 mL) and washed with ethyl acetate
(10 mL ꢂ 3) and the aqueous solution was then neutralized to pH
5 by slow addition of glacial acetic acid. Then the product precipi-
tated (crystallized) out to give 11b–11g.
3.1.2.8. 3-(2,6-Dichlorophenyl)-5-methyl-N-o-tolylisoxazole-4-
carboxamide (7h). Yield 79%; 1H NMR (CDCl3) d 0.83 (m, 2H),
1.04 (m, 1H), 1.19–1.32 (m, 4H), 1.39 (m, 1H), 2.02 (m, 2H), 2.73
(s, 3H), 3.74 (m, 1H), 5.02 (m, 1H), 7.39 (dd, 1H, J = 6.4 Hz,
9.6 Hz), 7.45 (m, 2H); 13C NMR (CDCl3) d 13.3, 24.1, 25.5, 32.5,
47.4, 112.0, 127.8, 128.7, 132.4, 136.3, 156.1, 159.9, 174.7.
HRMS-ESI (+): Calcd for C17H19N2O2Cl2: 353.0824. Found:
353.0838 [M+H]+.
3.1.3.1. 2-Mercaptobenzoxazole (11b). Yield 52%; 1H NMR
(DMSO-d6) d 7.29 (m, 3H), 7.53 (d, 1H, J = 7.6 Hz), 13.90 (br s,
1H); 13C NMR (DMSO-d6) d 110.0, 110.5, 123.7, 125.1, 131.2,
148.1, 180.1. HRMS-ESI (+): Calcd for C7H6NOS: 152.0170. Found:
152.0170 [M+H]+.
3.1.2.9. N-(4-Bromo-3-methylphenyl)-3-(2,6-dichlorophenyl)-
5-methylisoxazole-4-carboxamide (7i). Yield 82%; 1H NMR
(CDCl3) d 0.28 (m, 2H), 0.65–0.70 (m, 4H), 0.81 (d, 2H, J = 6.8 Hz),
0.94 (m, 2H), 1.16–1.25 (m, 2H), 1.31–1.44 (m, 4H), 1.49–1.58
(m, 4H), 1.75 (m, 2H), 3.63 (m, 1H), 4.10 (m, 1H), 7.39–7.51 (m,
5H); 13C NMR (CDCl3) d 13.2, 13.4, 22.2, 22.3, 29.6, 29.8, 31.5,
31.9, 32.9, 33.6, 44.3, 48.3, 111.8, 112.0, 127.7, 127.9, 128.7,
128.9, 132.3, 132.6, 136.2, 136.4, 156.0, 156.2, 159.8, 160.0,
174.5, 175.3. HRMS-ESI (+): Calcd for C18H21N2O2Cl2: 367.0980.
Found: 367.0991 [M+H]+.
3.1.3.2. 2-Mercapto-5-methylbenzoxazole (11c). Yield 45%; 1H
NMR (DMSO-d6) d 2.37 (s, 3H), 7.05 (m, 2H), 7.34 (m, 1H), 13.73
(br s, 1H); 13C NMR (DMSO-d6) d 20.8, 109.4, 110.4, 124.2, 131.2,
134.7, 146.3, 180.2. HRMS-ESI (+): Calcd for C8H8NOS: 166.0327.
Found: 166.0333 [M+H]+.
3.1.3.3. 2-Mercapto-6-methylbenzoxazole (11d). Yield 64%; 1H
NMR (DMSO-d6) d 2.39 (s, 3H), 7.10 (d, 1H, J = 7.6 Hz), 7.16 (t,
1H, J = 7.6 Hz), 7.31 (d, 1H, J = 7.6 Hz), 13.95 (br s, 1H); 13C NMR
(DMSO-d6) d 16.1, 107.2, 121.1, 123.6, 126.0, 130.4, 147.9, 180.1.
HRMS-ESI (+): Calcd for C8H8NOS: 166.0327. Found: 166.0333
[M+H]+.
3.1.2.10. (3-(2,6-Dichlorophenyl)-5-methylisoxazol-4-yl)(thio-
morpholino)methanone (7j). Yield 85%; 1H NMR (CDCl3) d 1.34
(br s, 4H), 1.52 (m, 2H), 2.56 (s, 3H), 3.38 (br s, 4H), 7.32 (m, 1H),
7.40 (m, 2H); 13C NMR (CDCl3) d 12.4, 24.4, 25.9, 113.6, 127.7,
128.4, 131.5, 135.9, 157.5, 161.6, 169.6. HRMS-ESI (+): Calcd for
C16H17N2O2Cl2: 339.0667. Found: 339.0668[M+H]+.
3.1.3.4. 2-Mercapto-3-nitrobenzoxazole (11e). Yield 14%; 1H
NMR (DMSO-d6) d 7.44 (t, 1H, J = 8.0 Hz), 7.91 (d, 1H, J = 8.0 Hz),
8.06 (d, 1H, J = 8.0 Hz); 13C NMR (DMSO-d6) d 115.8, 119.8, 123.4,
128.0, 131.4, 149.8, 181.7. HRMS-ESI (+): Calcd for C7H5N2O3S:
197.0021. Found: 197.0013 [M+H]+.
3.1.2.11. 3-(2,6-Dichlorophenyl)-N-(2-fluorophenyl)-5-methylis-
oxazole-4-carboxamide (7k). Yield 84%; 1H NMR (CDCl3) d 2.52
(s, 3H), 3.36 (br s, 8H), 7.30 (dd, 1H, J = 6.4 Hz, 9.6 Hz), 7.37 (m,
2H); 13C NMR (CDCl3) d 12.6, 66.7, 112.8, 127.4, 128.6, 131.8,
135.9, 157.3, 161.9, 170.4. HRMS-ESI (+): Calcd for C15H15N2O3Cl2:
341.0460. Found: 341.0466 [M+H]+.
3.1.3.5. 2-Mercapto-5-nitrobenzoxazole (11f). Yield 25%; 1H
NMR (DMSO-d6) d 7.17 (d, 1H, J = 8.8 Hz), 7.95 (d, 1H, J = 2.4 Hz),
7.99 (dd, 1H, J = 2.4 Hz, 8.8 Hz); 13C NMR (DMSO-d6) d 101.8,
112.3, 119.8, 139.7, 150.3, 153.2, 188.4. HRMS-ESI (+): Calcd for
C7H5N2O3S: 197.0021. Found: 197.0018 [M+H]+.
3.1.2.12. N-(3-Chloro-4-fluorophenyl)-3-(2,6-dichlorophenyl)-
5-methylisoxazole-4-carboxamide (7l). Yield 72%; 1H NMR
(CDCl3) d 2.30 (br s, 4H), 2.52 (s, 3H), 3.62 (br s, 4H), 7.30 (dd,
1H, J = 6.4 Hz, 9.6 Hz), 7.37 (m, 2H); 13C NMR (CDCl3) d 12.6,
27.8, 113.0, 127.4, 128.6, 131.9, 135.9, 157.2, 162.2, 170.4.
3.1.3.6. 2-Mercapto-5-chlorobenzoxazole (11g). Yield 36%; 1H
NMR (DMSO-d6) d 7.30 (d, 2H, J = 7.6 Hz), 7.53 (d, 1H, J = 8.0 Hz),
14.04 (br s, 1H); 13C NMR (DMSO-d6) d 110.3, 111.2, 123.5, 129.3,

W. Chen et al. / Bioorg. Med. Chem. 18 (2010) 1617–1625
1623
132.6, 147.0, 180.8. HRMS-ESI (+): Calcd for C7H5NOSCl: 185.9780.
Found: 185.9789 [M+H]+.
176.2. HRMS-ESI (+): Calcd for C17H12N3OS: 306.0701. Found:
306.0714 [M+H]+.
3.1.4. General procedures for the preparation of 2-thiouracils
14a–14i
3.1.4.9. 5-Cyano-6-(1-naphthyl)-2-thiouracil (14i). Yield 22%;
1H NMR (DMSO-d6) d 7.64 (m, 3H), 7.74 (d, 1H, J = 6.8 Hz), 7.99
(dd, 1H, J = 6.0 Hz, 6.4 Hz), 8.06 (dd, 1H, J = 6.0 Hz, 6.4 Hz,), 8.15
(d, 1H, J = 8.4 Hz), 13.11 (s, 1H), 13.46 (br s, 1H); 13C NMR
(DMSO-d6) d 93.1, 114.5, 124.7, 125.2, 126.8, 127.2, 127.5, 128.3,
128.5, 129.5, 131.2, 132.8, 158.7, 161.1, 176.8. HRMS-ESI (+): Calcd
for C15H10N3OS: 280.0545. Found: 280.0554 [M+H]+.
To a solution of an aldehyde (RCHO, 10 mmol), ethyl cyanoace-
tate (1.0 mL, 10 mmol), and thiourea (0.76 g, 10 mmol) in absolute
ethanol (50 mL) was added piperidine (2.0 mL, 20 mmol); the mix-
ture was heated under reflux overnight and then cooled to room
temperature. The precipitate was dissolved in 0.5 M NaOH
(20 mL) and washed with ethyl acetate (10 mL ꢂ 3). The aqueous
solution was then neutralized to pH 2 by slow addition of 1 M
HCl. Then the product precipitated (crystallized) out to give 14a–
14i.
3.1.5. General procedure for the preparation of 2,20-(
a
,
a0
a0
-
xylene)bis(sulfanediyl)bisbenzothiazole (15a), 2,20-(
a,
-
xylene)bis(sulfanediyl)bisbenzoxazole (15b–g), and 2,20-(
xylene)bis(sulfanediyl)bis-4-oxopyrimidine (16a–i)
a
, -
a0
3.1.4.1. 5-Cyano-6-phenyl-2-thiouracil (14a). Yield 67%; 1H
NMR (DMSO-d6) d 7.62 (m, 5H), 13.19 (s, 1H), 13.32 (br s, 1H);
13C NMR (DMSO-d6) d 90.2, 113.8, 128.2, 128.6, 132.0, 158.2,
160.2, 176.0. MS-ESI (+): 252.0 [M+Na]+.
To a solution of the 2-mercaptobenzothiazole, 2-mercaptobenz-
oxazole, or 2-thiouracil derivatives (11a–g or 10a–h, 0.1 mmol)
and
a,
a0-xylenedibromide (12 mg, 0.045 mmol) in acetonitrile
(2.5 mL) was added K2CO3 (42 mg, 0.3 mmol). The mixture was
heated under reflux overnight and then cooled to room tempera-
ture. The liquid was removed on a rotavapor and the residue was
washed with 0.5 M NaOH (20 mL). Then the white solid residue
was dried in vacuum oven at 40 °C overnight to give 15a–15g or
16a–16h.
3.1.4.2. 5-Cyano-6-(3-tolyl)-2-thiouracil (14b). Yield 31%; 1H
NMR (DMSO-d6) d 2.39 (s, 3H), 7.46 (m, 4H), 13.17 (s, 1H), 13.26
(br s, 1H); 13C NMR (DMSO-d6) d 20.9, 90.6, 114.7, 125.9, 128.4,
129.1, 129.2, 132.8, 137.9, 158.5, 160.9, 176.2. HRMS-ESI (+): Calcd
for C12H10N3OS: 244.0545. Found: 244.0555 [M+H]+.
3.1.5.1. 2,20-( a0-Xylene)bis(sulfanediyl)bis-(6-chlorobenzothi-
a,
3.1.4.3. 5-Cyano-6-(4-tolyl)-2-thiouracil (14c). Yield 43%; 1H
NMR (DMSO-d6) d 2.45 (s, 3H), 7.41 (d, 2H, J = 7.6 Hz), 7.61 (d,
2H, J = 8.4 Hz); 13C NMR (DMSO-d6) d 12.2, 81.7, 105.9, 118.4,
120.1, 121.2, 135.3, 150.9, 153.2, 168.2. HRMS-ESI (+): Calcd for
C13H12N3OS: 258.0701. Found: 258.0702 [M+H]+.
azole) (15a). Yield 84%; 1H NMR (DMSO-d6) d 4.62 (s, 4H), 7.45 (s,
4H), 7.47 (dd, 2H, J = 2.0 Hz, 8.4 Hz), 7.84 (d, 2H, J = 8.4 Hz), 8.11 (d,
2H, J = 2.0 Hz); 13C NMR (DMSO-d6) d 36.3, 120.8, 121.7, 126.2,
128.7, 135.4, 135.9, 151.0, 166.6. HRMS-ESI (+): Calcd for
C22H15N2S4Cl2: 504.9495. Found: 504.9499 [M+H]+.
3.1.4.4. 5-Cyano-6-(4-ethylphenyl)-2-thiouracil
(14d). Yield
25%; 1H NMR (DMSO-d6) d 1.22 (t, 3H, J = 7.6 Hz), 2.71 (q, 2H,
J = 7.6 Hz), 7.42 (d, 2H, J = 8.0 Hz), 7.61 (d, 2H, J = 8.0 Hz), 13.15
(br s, 1H); 13C NMR (DMSO-d6) d 15.2, 28.1, 90.4, 114.8, 126.6,
127.9, 128.9, 148.6, 158.5, 160.9, 176.2. HRMS-ESI (+): Calcd for
C14H14N3OS: 272.0858. Found: 272.0867 [M+H]+.
3.1.5.2. 2,20-( a0-Xylene)bis(sulfanediyl)bis(benzoxazole)
a,
(15b). Yield 35%; 1H NMR (DMSO-d6) d 4.60 (s, 4H), 7.34 (m, 4H),
7.49 (m, 4H), 7.65 (m, 4H); 13C NMR (DMSO-d6) d 35.1, 110.2,
118.3, 124.3, 124.6, 129.2, 136.1, 141.2, 151.3, 163.8. HRMS-ESI
(+): Calcd for C22H17N2O2S2: 405.0731. Found: 405.0732 [M+H]+.
3.1.5.3. 2,20-( a0-Xylene)bis(sulfanediyl)bis-(5-methylbenzox-
a,
3.1.4.5. 5-Cyano-6-(4-isopropylphenyl)-2-thiouracil
(14e). Yield 37%; 1H NMR (DMSO-d6) d 1.24 (d, 6H, J = 6.8 Hz), 3.00
(septet, 1H, J = 6.8 Hz), 7.46 (d, 2H, J = 8.0 Hz), 7.62 (d, 2H,
J = 8.0 Hz), 13.15 (br s, 2H); 13C NMR (DMSO-d6) d 23.6, 33.5,
90.3, 114.9, 126.5, 126.7, 129.0, 153.1, 158.6, 160.8, 176.2.
HRMS-ESI (+): Calcd for C14H14N3OS: 272.0858. Found: 272.0867
[M+H]+.
azole) (15c). Yield 57%; 1H NMR (DMSO-d6) d 2.40 (s, 6H), 4.57
(s, 4H), 7.12 (d, 2H, J = 8.0 Hz), 7.46 (m, 8H); 13C NMR (DMSO-d6)
d 20.9, 35.1, 109.6, 118.3, 125.1, 129.2, 134.0, 136.2, 141.4, 149.5,
163.6. HRMS-ESI (+): Calcd for C24H21N2O2S2: 433.1044. Found:
433.1041 [M+H]+.
3.1.5.4. 2,20-( a0-Xylene)bis(sulfanediyl)bis-(6-methylbenzox-
a,
azole) (15d). Yield 86%; 1H NMR (DMSO-d6) d 2.49 (s, 6H), 4.59
(s, 4H), 7.18 (m, 4H), 7.43 (d, 2H, J = 7.6 Hz), 7.49 (s, 4H); 13C
NMR (DMSO-d6) d 16.0, 35.2, 107.5, 124.0, 125.1, 128.3, 129.3,
136.2, 140.4, 151.0, 162.6. HRMS-ESI (+): Calcd for C24H21N2O2S2:
433.1044. Found: 433.1050 [M+H]+.
3.1.4.6. 5-Cyano-6-(4-methoxyphenyl)-2-thiouracil (14f). Yield
21%; 1H NMR (DMSO-d6) d 3.86 (s, 3H), 7.12 (d, 2H, J = 8.8 Hz),
7.68 (d, 2H, J = 8.8 Hz), 13.13 (br s, 2H); 13C NMR (DMSO-d6) d
55.7, 89.9, 114.0, 115.2, 121.1, 131.0, 158.8, 160.6, 162.5, 176.3.
HRMS-ESI (+): Calcd for C12H10N3O2S: 260.0494. Found: 260.0496
[M+H]+.
3.1.5.5. 2,20-( a0-Xylene)bis(sulfanediyl)bis-(4-nitrobenzoxaz-
a,
ole) (15e). Yield 61%; 1H NMR (DMSO-d6) d 4.66 (s, 4H), 7.50 (t,
4H, J = 8.0 Hz), 7.52 (s, 4H), 8.01 (d, 2H, J = 8.0 Hz), 8.08 (d, 2H,
J = 8.0 Hz); 13C NMR (DMSO-d6) d 35.3, 115.6, 119.8, 123.5, 128.8,
135.4, 136.9, 152.6, 168.1. HRMS-ESI (+): Calcd for C22H15N4O6S2:
495.0433. Found: 495.0438 [M+H]+.
3.1.4.7. 5-Cyano-6-(4-bromophenyl)-2-thiouracil (14g). Yield
39%; 1H NMR (DMSO-d6) d 7.63 (d, 2H, J = 8.4 Hz), 7.80 (d, 2H,
J = 8.4 Hz), 13.21 (s, 1H), 13.37 (br s, 1H); 13C NMR (DMSO-d6) d
91.1, 114.6, 125.9, 128.5, 130.9, 131.6, 158.4, 160.0, 176.2.
HRMS-ESI (+): Calcd for C11H7N3OSBr: 307.9493. Found:
307.9504 [M+H]+.
3.1.5.6. 2,20-( a0-Xylene)bis(sulfanediyl)bis-(6-nitrobenzoxaz-
a,
ole) (15f). Yield 81%; 1H NMR (DMSO-d6) d 4.67 (s, 4H), 7.53 (s,
4H), 7.85 (d, 2H, J = 8.4 Hz), 8.27 (dd, 2H, J = 2.0 Hz, 8.4 Hz), 8.62
(d, 2H, J = 2.0 Hz); 13C NMR (DMSO-d6) d 35.4, 106.9, 118.2,
121.0, 129.4, 135.9, 143.9, 146.6, 150.6, 170.0. HRMS-ESI (+): Calcd
for C22H15N4O6S2: 495.0433. Found: 495.0431 [M+H]+.
3.1.4.8. 5-Cyano-6-(biphenyl-4-yl)-2-thiouracil
(14h). Yield
75%; 1H NMR (DMSO-d6) d 7.45 (t, 1H, J = 7.2 Hz), 7.53 (t, 2H,
J = 7.2 Hz), 7.78 (d, 4H, J = 8.4 Hz), 7.89 (d, 2H, J = 8.4 Hz) 13.19 (s,
1H), 13.35 (br s, 1H); 13C NMR (DMSO-d6) d 90.6, 114.8, 126.6,
127.0, 128.1, 128.4, 129.2, 129.6, 138.7, 143.7, 158.5, 160.5,

1624
W. Chen et al. / Bioorg. Med. Chem. 18 (2010) 1617–1625
3.1.5.7. 2,20-(
a,
a0-Xylene)bis(sulfanediyl)bis-(5-chlorobenzoxa-
J = 7.6 Hz), 7.75 (m, 8H), 7.88 (d, 4H, J = 8.4 Hz); 13C NMR (DMSO-
d6) d 33.7, 88.8, 120.3, 126.3, 126.8, 127.8, 128.7, 128.9, 129.0,
136.7, 137.4, 139.5, 141.3, 166.4, 170.4, 171.8. HRMS-ESI (ꢃ): Calcd
for C42H27N6O2S2: 711.1637. Found: 711.1661 [MꢃH]ꢃ.
zole) (15g). Yield 49%; 1H NMR (DMSO-d6) d 4.60 (s, 4H), 7.36 (dd,
2H, J = 2.0 Hz, 8.8 Hz), 7.48 (s, 4H), 7.67 (d, 2H, J = 8.8 Hz), 7.76 (d,
2H, J = 2.0 Hz); 13C NMR (DMSO-d6) d 35.2, 111.5, 118.1, 124.3,
129.0, 129.3, 136.0, 142.5, 150.1, 165.9. HRMS-ESI (+): Calcd for
C22H15N2O2S2Cl2: 472.9952. Found: 472.9974 [M+H]+.
3.1.5.16. 2,20-( a0-Xylene)bis(sulfanediyl)bis-(6-(1-naphthyl)-
a,
5-cyano-4-oxopyrimidine) (16i). Yield 92%; 1H NMR (CD3OD) d
4.37 (s, 4H), 7.32 (s, 4H), 7.44–7.57 (m, 8H), 7.74 (d, 2H,
J = 8.0 Hz), 7.91 (d, 2H, J = 8.2 Hz), 7.96 (dd, 2H, J = 2.0 Hz,
7.0 Hz); 13C NMR (CD3OD) d 35.7, 94.1, 119.1, 126.2, 126.5, 127.4,
127.8, 127.9, 129.5, 130.3, 130.9, 132.0, 135.2, 136.6, 138.5,
171.6, 174.0, 175.0. HRMS-ESI (ꢃ): Calcd for C38H23N6O2S2:
659.1324. Found: 659.1343 [MꢃH]ꢃ.
3.1.5.8. 2,20-( a0-Xylene)bis(sulfanediyl)bis-(6-phenyl-5-
a,
cyano-4-oxopyrimidine) (16a). Yield 80%; 1H NMR (DMSO-d6) d
4.26 (s, 4H), 7.31 (s, 4H), 7.44 (m, 6H), 7.77 (m, 4H); 13C NMR
(DMSO-d6) d 33.6, 88.8, 119.9, 127.8, 127.9, 128.6, 129.3, 137.2,
137.7, 166.8, 170.3, 171.5. HRMS-ESI (ꢃ): Calcd for C30H19N6O2S2:
559.1011. Found: 559.0989 [MꢃH]ꢃ.
3.1.5.9. 2,20-( a0-Xylene)bis(sulfanediyl)bis-(6-(3-tolyl)-5-
a,
3.1.6. General procedure for the preparation of S-benzyl-2-
thiouracils (17d,e,g–i)
cyano-4-oxopyrimidine) (16b). Yield 62%; 1H NMR (DMSO-d6) d
2.35 (s, 6H), 4.24 (s, 4H), 7.31 (m, 8H), 7.54 (s, 4H); 13C NMR
(DMSO-d6) d 21.0, 33.6, 88.9, 120.1, 125.3, 127.9, 128.6, 128.8,
130.2, 137.2, 137.4, 137.8, 167.2, 170.5, 171.6. HRMS-ESI (ꢃ): Calcd
for C32H23N6O2S2: 587.1324. Found: 587.1348 [MꢃH]ꢃ.
To a solution of the 2-thiouracil derivatives (10d,e,g–i, 2 mmol)
and benzylchloride (253 mg, 2 mmol) in acetonitrile (10 mL) was
added K2CO3 (829 mg, 6 mmol). The mixture was heated under re-
flux for 8 h and then cooled to room temperature. The liquid was
removed on a rotavapor, and the residue was washed by H2O
(20 mL). Then the solid was dried in a vacuum oven at 40 °C over-
night to give 17d,e,g–i.
3.1.5.10. 2,20-( a0-Xylene)bis(sulfanediyl)bis-(6-(4-tolyl)-5-
a,
cyano-4-oxopyrimidine) (16c). Yield 80%; 1H NMR (DMSO-d6) d
2.36 (s, 6H), 4.25 (s, 4H), 7.26 (d, 4H, J = 8.0 Hz), 7.32 (s, 4H), 7.68
(d, 4H, J = 8.0 Hz); 13C NMR (DMSO-d6) d 21.0, 33.7, 88.6, 120.2,
128.1, 128.6, 128.9, 134.9, 137.4, 139.4, 166.9, 170.7, 171.7.
HRMS-ESI (ꢃ): Calcd for C32H23N6O2S2: 587.1324. Found:
587.1306 [MꢃH]ꢃ.
3.1.6.1. S-Benzyl-5-cyano-6-(4-ethylphenyl)-2-thiouracil
(17d). Yield 28%; 1H NMR (DMSO-d6) d 1.27 (t, 3H, J = 7.6 Hz), 2.71
(q, 2H, J = 7.6 Hz), 4.40 (s, 2H), 7.21 (m, 1H), 7.28 (m, 4H), 7.41 (d,
2H, J = 7.2 Hz), 7.74(d, 2H, J = 8.4 Hz); 13C NMR (DMSO-d6) d 16.0,
29.8, 36.0, 90.6, 120.1, 128, 128.8, 129.2, 129.4, 129.8, 130.1,
136.0, 139.8, 148.2, 170.2, 174.9. HRMS-ESI (+): Calcd for
C20H18N3OS: 348.1171. Found: 348.1185 [M+H]+.
3.1.5.11. 2,20-( a0-Xylene)bis(sulfanediyl)bis-(6-(4-ethylphe-
a,
nyl)-5-cyano-4-oxopyrimidine) (16d). Yield 96%; 1H NMR
(DMSO-d6) d 1.21 (t, 6H, J = 7.6 Hz), 2.65 (q, 4H, J = 7.6 Hz), 4.26
(s, 4H), 7.29 (d, 4H, J = 8.0 Hz), 7.32 (s, 4H), 7.70 (d, 4H,
J = 8.0 Hz); 13C NMR (DMSO-d6) d 15.5, 28.1, 33.7, 88.6, 120.3,
127.5, 128.2, 128.9, 135.2, 137.4, 145.6, 166.9, 170.7, 171.7.
HRMS-ESI (ꢃ): Calcd for C34H27N6O2S2: 615.1637. Found:
615.1613 [MꢃH]ꢃ.
3.1.6.2. S-Benzyl-5-cyano-6-(4-isopropylphenyl)-2-thiouracil
(17e). Yield 40%; 1H NMR (DMSO-d6) d 1.23 (d, 6H, J = 6.8 Hz), 2.94
(septet, 1H, J = 6.8 Hz), 4.30 (s, 2H), 7.22 (t, 1H, J = 6.8 Hz), 7.31 (m,
4H), 7.40 (d, 2H, J = 7.2 Hz), 7.73 (d, 2H, J = 7.6 Hz); 13C NMR
(DMSO-d6) d 23.7, 33.3, 33.8, 88.7, 120.3, 126.0, 126.7, 128.2,
128.3, 128.8, 135.3, 139.0, 150.2, 166.8, 170.5, 171.5. HRMS-ESI
(+): Calcd for C21H20N3OS: 362.1327. Found: 362.1335 [M+H]+.
3.1.5.12. 2,20-( a0-Xylene)bis(sulfanediyl)bis-(6-(4-isopropyl-
a,
phenyl)-5-cyano-4-oxopyrimidine) (16e). Yield 87%; 1H NMR
(DMSO-d6) d 1.25 (d, 12H, J = 7.2 Hz), 2.98 (septet, 2H, J = 7.2 Hz),
4.52 (s, 4H), 7.36 (s, 4H), 7.38 (d, 4H, J = 8.4 Hz), 7.87 (d, 4H,
J = 8.0 Hz); 13C NMR (DMSO-d6) d 22.7, 32.7, 33.7, 91.8, 114.9,
125.8, 128.2, 128.4, 132.2, 135.3, 152.1, 161.1, 165.3, 166.4.
HRMS-ESI (ꢃ): Calcd for C36H31N6O2S2: 643.1950. Found:
643.1943 [MꢃH]ꢃ.
3.1.6.3. S-Benzyl-5-cyano-6-(4-bromophenyl)-2-thiouracil
(17g). Yield 33%; 1H NMR (DMSO-d6) d 4.27 (s, 2H), 7.22 (t, 1H,
J = 7.2 Hz), 7.29 (t, 2H, J = 7.2 Hz), 7.39 (d, 2H, J = 7.2 Hz), 7.67 (d,
2H, J = 8.4 Hz), 7.71 (d, 2H, J = 8.4 Hz); 13C NMR (DMSO-d6) d
33.8, 88.9,120.0, 123.2, 126.7, 128.3, 128.9, 130.2, 131.1, 136.9,
139.0 165.8, 170.1, 171.8. HRMS-ESI (+): Calcd for C18H13N3OSBr:
397.9963. Found: 397.9950 [M+H]+.
3.1.5.13. 2,20-( a0-Xylene)bis(sulfanediyl)bis-(6-(4-methoxy-
a,
phenyl)-5-cyano-4-oxopyrimidine) (16f). Yield 46%; 1H NMR
(DMSO-d6) d 3.81(s, 6H), 4.28 (s, 4H), 6.97 (d, 4H, J = 8.8 Hz), 7.30
(s, 4H), 7.82 (d, 4H, J = 8.8 Hz); 13C NMR (DMSO-d6) d 33.7, 55.3,
88.2, 113.5, 120.4, 128.9, 129.8, 130.0, 137.4, 160.6, 166.3, 170.6,
171.4. HRMS-ESI (ꢃ): Calcd for C32H23N6O4S2: 619.1222. Found:
619.1230 [MꢃH]ꢃ.
3.1.6.4. S-Benzyl-5-cyano-6-(biphenyl-4-yl)-2-thiouracil
(17h). Yield 37%; 1H NMR (DMSO-d6) d 4.32 (s, 2H), 7.23 (t, 1H,
J = 7.6 Hz), 7.31 (t, 2H, J = 7.6 Hz), 7.40 (m, 3H), 7.50 (t, 2H,
J = 7.6 Hz), 7.74 (d, 2H, J = 8.0 Hz), 7.77 (d, 2H, J = 8.4 Hz), 7.90 (d,
2H, J = 8.0 Hz); 13C NMR (DMSO-d6) d 33.8, 89.1, 126.4, 126.8,
126.8, 127.9, 128.3, 128.8, 128.9, 129.0, 136.6, 139.0, 139.4,
141.4, 166.4, 169.7, 171.3. HRMS-ESI (+): Calcd for C24H18N3OS:
396.1171. Found: 396.1187 [M+H]+.
3.1.5.14. 2,20-( a0-Xylene)bis(sulfanediyl)bis-(6-(4-bromophe-
a,
nyl)-5-cyano-4-oxopyrimidine) (16g). Yield 95%; 1H NMR
(DMSO-d6) d 4.25 (s, 4H), 7.31 (s, 4H), 7.67 (d, 4H, J = 8.4 Hz),
7.72 (d, 4H, J = 8.8 Hz); 13C NMR (DMSO-d6) d 33.7, 88.9, 119.9,
123.3, 128.9, 130.2, 131.1, 136.8, 137.3, 165.9, 170.2, 171.8.
HRMS-ESI (ꢃ): Calcd for C30H18Br2N6O2S2: 714.9221. Found:
714.9213 [MꢃH]ꢃ.
3.1.6.5. S-Benzyl-5-cyano-6-(1-naphthyl)-2-thiouracil
(17i). Yield 43%; 1H NMR (DMSO-d6) d 4.26 (s, 2H), 7.23 (t, 1H,
J = 7.2 Hz), 7.29 (t, 2H, J = 7.2 Hz), 7.39 (d, 2H, J = 7.2 Hz), 7.55 (m,
5H), 7.78 (d, 1H, J = 8.0 Hz), 7.99 (t, 2H, J = 6.8 Hz); 13C NMR
(DMSO-d6) d 33.8, 92.4, 119.3, 125.2, 125.4, 126.0, 126.2, 126.4,
126.8, 128.2, 128.3, 128.9, 130.1, 133.1, 135.9, 138.9, 168.7,
167.6, 171.4. HRMS-ESI (+): Calcd for C22H16N3OS: 370.1014.
Found: 370.1015 [M+H]+.
3.1.5.15. 2,20-( a0-Xylene)bis(sulfanediyl)bis-(6-(biphenyl-4-
a,
yl)-5-cyano-4-oxopyrimidine) (16h). Yield 68%; 1H NMR (DMSO-
d6) d 4.28 (s, 4H), 7.35 (s, 4H), 7.40 (t, 2H, J = 7.2 Hz), 7.49 (t, 4H,

W. Chen et al. / Bioorg. Med. Chem. 18 (2010) 1617–1625
1625
3.2. Biological evaluation
formed on URSA, a 160-processor computer based on the Power5+
processor and IBM0s P series architecture.
3.2.1. General in vitro biological methods
EcN68, the N-terminal fragment of SecA from E. coli without the
C-terminal regulatory domain, and EcSecA, the full length SecA
from E. coli, were over-expressed from pIMBB-821 and pT7-SecA,22
respectively, and purified as described.23,24 EcN68 was used for
screening because it has higher intrinsic activity and is more sen-
sitive to inhibitors.
Acknowledgments
We gratefully acknowledge the financial support by the Georgia
Research Alliance, Georgia Cancer Coalition, NIH Grant GM34766,
and the Molecular Basis of Disease Program at GSU.
All potential inhibitors were dissolved in 100% DMSO. The ATP-
ase activity was determined by the release of phosphate (Pi) de-
Supplementary data
tected by malachite green as described3 in
a
modified
procedure15 and in the presence of 10% DMSO. Inhibitory effect
was determined by the percentage of the remaining ATPase activ-
ity as compare to the controls without test compounds. Briefly,
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.12.074.
50
N68 or 5
l
L reaction mixture was prepared so that it contained 2.25
lg
References and notes
lg SecA, 2 mM ATP, 50 mM Tris–HCl (pH7.6), 20 mM
KCl, 20 mM NH4Cl, 1 mM DTT, and 2 mM Mg(OAc)2. Reactions took
place at 40 °C for 20 min (for N68) or 40 min (for SecA) then were
stopped by adding 800 lL of malachite green and then 100 lL of
34% citric acid within 1 min. The mixtures were incubated at room
temperature for 40 min and then the absorption at 660 nm was
measured. All assays were done at least in triplicate, and the re-
sults were presented as bar graphs with standard error of the
mean.
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of about 30
IC50 of 100
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l
l
M. However, upon more rigorous studies, 2 was found to have
M. 1 showed similar inhibition activities as 2, but started having
M.
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8
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out on a Xeon-based Linux workstation. Molecular mechanics cal-
culations and molecular dynamics simulations (AMBER 8) were per-
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