Identification and validation of small molecule modulators of the NusB-NusE interaction

Article abstract of DOI:10.1016/j.bmcl.2016.11.091

Formation of highly possessive antitermination complexes is crucial for the efficient transcription of stable RNA in all bacteria. A key step in the formation of these complexes is the protein-protein interaction (PPI) between N-utilisation substances (Nu

Full text of DOI:10.1016/j.bmcl.2016.11.091

Bioorganic & Medicinal Chemistry Letters 27 (2017) 162–167
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification and validation of small molecule modulators
of the NusB-NusE interaction
Peter J. Cossar a,3, Cong Ma b,2,3, Christopher P. Gordon a,1, Joseph I. Ambrus a, Peter J. Lewis b,
Adam McCluskey a,
⇑
a Chemistry, School of Environmental and Life Sciences, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia
b Biology, Centre for Chemical Biology, School of Environmental and Life Sciences, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Formation of highly possessive antitermination complexes is crucial for the efficient transcription of
stable RNA in all bacteria. A key step in the formation of these complexes is the protein-protein interac-
tion (PPI) between N-utilisation substances (Nus) B and E and thus this PPI offers a novel target for a new
antibiotic class. A pharmacophore developed via a secondary structure epitope approach was utilised to
perform an in silico screen of the mini-Maybridge library (56,000 compounds) which identified 25 hits of
which five compounds were synthetically tractable leads. Here we report the synthesis of these five leads
and their biological evaluation as potential inhibitors of the NusB-NusE PPI. Two chemically diverse scaf-
folds were identified to be low micro molar potent PPI inhibitors, with compound (4,6-bis(20,40,3.4
Received 24 September 2016
Revised 29 November 2016
Accepted 30 November 2016
Available online 1 December 2016
Keywords:
Protein-protein interaction
NusB-NusE
Antibiotic
Pharmacophore
In silico screening
tetramethoxyphenyl))pyrimidine-2-sulphonamido-N-4-acetamide
1
and N,N0-[1,4-butanediylbis(oxy-
M, respectively. These inhi-
4,1-phenylene)]bis(N-ethyl)urea 3 exhibiting IC50 values of 6.1 M and 19.8
l
l
bitors were also shown to be moderate inhibitors of Gram-positive Bacillus subtilis and Gram-negative
Escherichia coli growth.
Ó 2016 Published by Elsevier Ltd.
Antibiotic resistance has evolved against all clinically approved
antibiotics.1–5 Exacerbating this problem is the withdrawal of all
major pharmaceutical companies from antibiotic research effec-
tively severing the traditional antibiotic drug development
pipeline.6,7 Of equal concern is that the majority of new antibiotics
are derivatives of existing drugs for which resistance rapidly arises
or is even pre-existing.8 Consequently, there is an urgent need to
develop new antibiotic classes which are not predisposed to the
development of drug resistance.3,4,6–13
The current arsenal of antibiotics typically target four
major processes within bacteria: a) cell wall/membrane
synthesis, b) translation, c) DNA replication and d) inhibition of
metabolism.1,2,14 Thus an underutilised target for antibiotic devel-
opment is the critical process of transcription, with only Rifamycin
and Fidaxomicin approved for limited clinical use as anti-transcrip-
tion targeted drugs.15 Transcription inhibition has the potential to
offer a number of new targets for antibiotic drug development, and
of particular interest are a number of critical protein-protein
interactions (PPIs) which are essential for transcription
regulation.16,17
There is a growing body of evidence suggesting that small mole-
cule inhibitors can be used to inhibit PPIs.18–21 These inhibitors
typically target a small area generally at the centre of an interface
which confers the essential binding interactions. These clusters of
amino acids are termed ‘‘hot spots”. The efficacy of PPI inhibition
can vary from micro- to pico-molar potent.22 We believed that this
approach could be utilised to develop a new class of antibiotics
which inhibit the formation of the antitermination complex. This
large nucleoprotein assembly is unique to bacteria and functions
to regulate the transcription of bacterial stable RNA (t- and
rRNA).23,24 An essential stage in the formation of this complex is
the PPI between N-utilisation substance (Nus) B and N-utilisation
substance E (NusB-NusE), which is responsible for initiation and
recruitment of other Nus proteins and RNA Polymerase to form
the antitermination complex.25–27
⇑
Corresponding author.
E-mail address: Adam.McCluskey@newcastle.edu.au (A. McCluskey).
Present address: Nanoscale Organization and Dynamics Group, School of Science
1
and Health, Western Sydney University, Penrith South DC, NSW, Australia.
The NusB-NusE interface is characterised by the
a1-helix of
2
Present address: Department of Applied Biology and Chemical Technology, The
NusE which occupies a pocket of NusB (Fig. 1). This interaction is
established by the amino acid residues H15, R16 and D19 of the
State Key Laboratory of Chirosciences, The Hong Kong Polytechnic University, Hung
Hom, Kowloon, Hong Kong Special Administrative Region.
3
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2016.11.091
0960-894X/Ó 2016 Published by Elsevier Ltd.

P.J. Cossar et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 162–167
163
PPI interface as a potentially druggable target. Building on this
we used a secondary structure epitope approach comprising a sin-
gle face of the a1-helix of NusE with critical hot spot residues H15,
R16 and D19 of the A. aeolicus NusB-NusE-boxA structure (PDB:
3R2C)29 to develop a NusB-NusE interaction pharmacophore (Sup-
plementary Data).
Our pharmacophore was developed from the partial sequence
alignment of
a1-helix NusE and two sequences which comprise
the NusB binding pocket. Sequence alignment of NusE and NusB
from Aquifex aeolicus, Bacillus subtilis, Escherichia coli, Haemophilus
influenza, Helicobacter pylori, Pseudomonas aeruginosa, Mycobac-
terium tuberculosis, Staphylococcus aureus and Streptococcus pneu-
moniae highlighted high sequence homology and conservation
across a range of bacterial species supportive of a broad spectrum
antibacterial target. Of particular note, the NusE
minimal sequence deviation, suggesting its essential role in
NusB-NusE PPI binding (Fig. 3A).
a1-helix showed
Fig. 1. The protein-protein interface of Aquifex aeolicus NusB-NusE (PDB ID 3R2C).29
Depicted in blue is the protein NusB, with essential residues Y18, E81 and R76
highlighted (sticks). Shown in green is the
a1-helix of NusE, with key amino acids
Having identified the conserved nature of these proteins, we
next compared the protein alignment with published NMR and
X-ray crystal structures of the NusB-NusE heterodimer in E. coli
(PDB: 3D3B) and A. aeolicus (PDB: 3R2C) to determine key amino
acid interactions.28,29,31 Our analysis identified the major hydrogen
bonding contributions as NusB E81 (E. coli E81)-NusE H15 (E. coli
H15), NusB Y16 (E. coli Y18)-NusE D19 (E. coli D19) and NusB
R76 (E. coli E75)-NusE R16 (E. coli R16). These data were consistent
with the hotspot mutation identified by Friedman et al.27 These
three key amino acid interactions were then superimposed as
hydrogen bond acceptor or hydrogen bond donor query features,
according to the characteristics of each amino acid, over a single
H15, D19 and R16 (sticks) interacting with the binding pocket of NusB.
a1-helix forming electrostatic links with residues Y18, R76 and
E81 of NusB. Combined, this pocket consists of eight hydrogen
bond interactions, five of which are considered essential for het-
erodimer formation.28,29
Significantly, the residues identified above are conserved across
many medically important bacterial strains including Staphyloccus
aureus, Streptococcus pneumoniae and Haemophilus influenza
(Fig. 2). Moreover, the importance of this interaction was demon-
strated by two point mutations in E. coli, nusE100 (R72G)30 and
nusB5 (Y18D);26 these mutations are buried in the interface and
directly disrupt protein-protein binding, which in turn impedes
antitermination complex formation. We hypothesised that by
face of
a1-helix of NusE using Discovery Studio (BIOVIA)
(Fig. 2b). This gave rise to a hydrogen bond acceptor (x = 18.197;
y = ꢀ30.736; z = 47.491) and two hydrogen bond donors
(x = 27.296; y = ꢀ21.920; z = 42.021 and x = 25.296; y = ꢀ23.413;
z = 48.000) regions. The features were used to define a central
point on the residue and a location constraint sphere with a radius
of 1.6 Å (based on a strong hydrogen bond interaction). In an effort
to minimise potential steric clashes, a total of 21 exclusion spheres
of 1.2 Å radius were installed and ultimately defined the shallow
binding groove of NusB.
mimicking the
a1-helix of NusE, in particular H15, R16 and D19,
we could competitively inhibit the NusB-NusE PPI, and develop a
platform for subsequent identification of small molecule modula-
tors of the interaction.
To support our hypothesis that targeting the NusB-NusE PPI
would prevent the assembly of this complex a 9-mer peptide, H-
YDHRLLDQS-NH2, was synthesised and screened as a potential
In total, the NusB-NusE pharmacophore comprised a hydrogen
bond acceptor, two hydrogen bond donors and 21 exclusion zones.
inhibitor. The 9-mer returned an IC50 of 71 6.2
the potential to inhibit this PPI and supporting the NusB-NusE
lM, confirming
Fig. 2. Protein sequence alignment of NusE spanning 17 species of bacteria. Black regions indicate conserved residues; grey regions, partial conservation; white region, no
residue conservation across species. The region highlighted by the red boundary represents the key NusE residues involved in binding to NusB: H15, R16 and D19.

164
P.J. Cossar et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 162–167
Fig. 3. A) The protein alignment of NusE spanning nine species of bacteria. Black regions indicate conserved residues; grey regions, partial conservation; and white regions, no
residue conservation across species. The red boxed section highlights the key NusE residues involved in binding to NusB. The coloured circles above the red-boxed section
denote the amino acids involved in the formation of hydrogen bond interactions where magenta = hydrogen bond donors (H15 and R16) and green = hydrogen bond acceptor
(D19); B) Schematic representation of the NusB-NusE pharmacophore features overlaid onto NusE protein, where the magenta and green spheres represents hydrogen bond
donors and hydrogen bond acceptors, respectively. Grey spheres represent pharmacophore exclusion zones.
O
HN
OH
H
H3CO
N
O
N
S
HN
O
O
H3CO
2
N
N
OCH3
OCH3
H3CO
H3CO
HO
1
3
OCH3
H
N
H
H
N
O
N
O
O
O
N
H
4
O
O
OH
N
H
N
H
OH
N
N
H
N
O
N
H
CF3
5
O2N
Fig. 4. Chemical structures of five synthetically tractable lead compounds from pharmacophore based in silico screening of the Maybridge 56,000 compound library.
Application of this pharmacophore as described by Yang et al. and
an in silico screen of the mini-Maybridge 56,000 compound library
identified 25 preliminary hits which were then subjected to further
energy minimisations.32 Remapping of these hits was followed by
manual inspection of the resulting conformations, which as a con-
sequence of poor pharmacophore overlap identified only five com-
pounds, that were also deemed readily synthetically accessible
(Fig. 4).32
PPI inhibitors. Pyrimidine 1 was accessed as per Scheme 1;33 Aldol
condensation of 3,4-dimethoxyacetophenone 6, and 2,4-
dimethoxybenzaldehyde 7 gave chalcone 8 (85%). Initial efforts
to cyclise 8 using literature approaches proved unsuccessful how-
ever,33,34 with sodium isopropoxide treatment of 8 with 4-amino-
N-(aminoiminomethylbenezenesulfonamide) resulting in a ꢁ4:1
mixture of the desired pyrimidine 9 and the unexpected dihy-
dropyrimidine 10. Attempts to oxidise this mixture with Jones
reagent failed to provide clean conversion to 9. However after a
number of optimisation studies pyridinium dichromate was iden-
To validate our pharmacophore and in silico screening hits, we
set about synthesising compounds 1–5 for screening as NusB-NusE

P.J. Cossar et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 162–167
165
O
OCH3
O
H3CO
H3CO
H3CO
O
i
H3CO
H3CO
+
OCH3
OCH3
6
7
8
ii
O
NH2
NH2
HN
O
O
O
S
iii, iv
S
N
O
O
HN
HN
O
+
S
N
HN
N
OCH3
N
NH
OCH3
N
OCH3
H3CO
H3CO
H3CO
H3CO
H3CO
OCH3 H3CO
OCH3
1
9
10
OCH3
Scheme 1. Reagents and conditions: i) 10% NaOH(aq), EtOH, r.t., 48 h, 63%; ii) 4-amino-N-(aminoiminomethylbenezenesulfonamide), sodium isopropoxide, isopropanol,
reflux, 16 h, (used as is without purification); iii) PDC, CH2Cl2, 16 h, 34% over 2 two-steps. iv) acetic anhydride, r.t., 50 °C, 16 h, 71%.
OH
H3CO
H3CO
OH
H
N
O
H3CO
H3CO
i
H
N
N
+
H2N
O
OCH3
O
OCH3
11
12
2
Scheme 2. Reagents and conditions: i) Ethanol, reflux, 16 h, 86%.
R
O2N
i
Br
O
+
Br
O
OH
13
R
14
R = NO2 (15)
R = NH2 (16)
ii
iii or iv
H
N
R
O
O
R
N
H
H
N
4, R =
3, R =
O
OH
Scheme 3. Reagents and conditions: i) Cs2CO3, KI, CH3CN, reflux, 16 h, 60%; ii) H-cubeÒ, Raney Ni, 1,4-dioxane, 50 °C, 50 bar, 1 mL.minꢀ1, circulated 5 times, 99%; iii) ethyl
isocyanate, THF, reflux, 16 h, 44%; iv) 2-acetoxybenzaldehyde, THF, r.t., 16 h, NaBH4 0 °C–RT, 30 min, 95%
tified as a reagent which afforded quantitative conversion of 10 to
9. Acetic anhydride mediated acetylation of 9 gave the lead
pyrimidine 1 (71%).
ment of 16 with ethyl isocyanate gave 3 (44%). Reductive amina-
tion of 16 with 2-acetoxybenzaldehyde followed by acetate
deprotection afforded 4 (98%).
Hydrazide 2 was obtained in a 86% yield by condensation
of 3,4,5-trimethoxybenzaldehyde 11 and 3-(4-hydroxyphenyl)
propanehydrazide 12 at ethanol reflux (Scheme 2).35
The symmetrical ethers 3 and 4 were generated from a common
intermediate 15, produced by coupling of 1,4-dibromobutane 13
and 4-nitrophenol 14 under modified Finkelstein conditions to
afford 15 (60%) (Scheme 3).36 Optimised H-Cube flow hydrogena-
tion conditions of 1 mL.minꢀ1, 50 bar H2, 50 °C over Raney Ni
furnished the desired diamine 16 in a quantitative yield and treat-
Racemic 5 was accessed through a convergent approach with
the epoxide 21 generated by coupling of 4-nitrophenol 14 with
( )-epichlorohydrin 19. The key amine 20 was accessed on treat-
ment of pyrimidine 17 with 1,2-diaminoethane 18. Nucleophilic
ring opening of epoxide 21 by 20 afforded racemic 5 (38%)
(Scheme 4).37
With screening hits 1–5 in hand, their ability to inhibit the
NusB-NusE PPI was examined at 25
lM compound concentration
in an ELISA against B. subtilis NusB and NusE proteins (Table 1).38

166
P.J. Cossar et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 162–167
CF3
CF3
i
NH2
N
+
N
H2N
NH2
N
N
H
CF3
N
Cl
17
18
14
20
N
OH
iii
H
N
O
N
N
H
NO2
NO2
5
ii
O
Cl
NO2
+
O
O
HO
19
21
Scheme 4. Reagents and conditions: i) 0 °C–RT, 1 h, 76%; ii) DMF, KI, Cs2CO3, 75 °C, 3 h, 55%; iii) EtOH, reflux, overnight, 38%.
9–44%. While the observed antibiotic activity poorly correlated
with the inhibition of the NusB-NusE PPI, these findings are consis-
tent with phenotypic studies where the physicochemical proper-
ties of the molecules can have an adverse effect on cellular (here
bacterial) uptake.
Table 1
The inhibition of the B. subtilis NusB-NusE PPI at 25
analogues 1–5, 8 and 9.
lM and the IC50 values of
Compound
Inhibition of nuB-nusE
interaction at 25 lM [%]
IC50 (lM)
1
2
3
4
5
8
9
88
5
6.1 1.2
n.d.a
19.8 1.7
210.0 2.1
–
Conclusions
52
45
n.ab,c
48
3
The inhibition of the NusB-NusE interaction was demonstrated
with the 9-mer, H-YDHRLLDQS-NH2, 71 6.2 lM potent inhibitor
of this interaction. The development and subsequent screening of
the Maybridge mini library with a pharmacophore based on the
NusB/NusE binding interface identified five synthetically tractable
hits. Synthesis of chemically diverse 1–5 and ELISA screening of the
NusB-NusE PPI revealed three compounds (1, 3 and 4) inhibited
n.d.
n.d.
a
b
c
n.d. = not determined.
n.a = not active.
Racemate.
NusB-NusE binding, with >50% binding efficiency at 25
dose response evaluation returned IC50 values of 6.1, 19.8 and
210 M, for 1, 3 and 4 respectively. Subsequent screening against
B. subtilis and E. coli showed moderate levels of antibacterial activ-
ity. Combined these findings validate both our pharmacophore
based approach and the inhibition of the NusB-NusE PPI as a valid
antibiotic drug development strategy.
Compounds 1, 3 and 4 represent the first reported, validated,
inhibitors of the NuB-NusE PPI and may allow the development
of new classes of antibiotics targeting bacterial transcription.
lM. Full
Table 2
l
Inhibition of B. subtilis and E. coli growth at 200 lM by analogues 1–5, 8 and 9 in
inhibition.
Compound
Bacterial growth inhibition at 200 lM [%]
Bacillus subtilis
Escherichia coli
1
2
3
9
44
21
10
17
n.a.
11
n.a.a
n.a.
19
4
5b
a
Acknowledgements
n.a. = not active.
Racemate.
b
This work was supported by: the Australian Cancer Research
Foundation, Ramaciotti Foundation, the Australian Research Coun-
cil, National Health and Medical Research Council (Australia), and
the University of Newcastle Early Career Research Grant (CM).
CPG is the recipient of an ARC DECRA fellowship. PJC acknowledges
the receipt of a University of Newcastle Postgraduate Research
Scholarship. The authors thank Mohammed K. Abdel-Hamid for
helpful discussions.
Pleasingly compounds, 1, 3 and 4 returned NusB-NusE PPI inhibi-
tion levels of 88%, 52% and 45% respectively with analogues 2
and 5 inactive. Full dose response evaluation of 1, 3 and 4 against
the NusB-NusE PPI returned IC50 values of 6.1, 19.8 and 210.0
respectively.
lM,
The advanced synthetic intermediates 8 and 9 were also sub-
jected to the NusB-NusE ELISA. The high level of activity observed
with 8 (48% at 25
cuous nature of the
l
M) was most probably a function of the promis-
a
A. Supplementary data
,b-unsaturated moiety.39 The low levels of
activity with 9, suggests a key role for the acetate moiety in the
inhibition of the NusB-NusE PPI (c.f. 1 at 88% versus 9 at 3%, at
Supplementary data (pharmacophore details, NMR, IR, mass
spectra and HPLC chromatograms) associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/j.
bmcl.2016.11.091.
25
l
M compound concentration).
The NusB-NusE PPI targeted analogues 1–5 were examined for
their potential inhibition of the ability to inhibit Gram positive B.
subtilis and Gram negative E. coli at 200 lM compound concentra-
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5 as a
white solid (0.23 g, 38%), 130–131 °C. 1H NMR
(400 MHz, DMSO-d6) d 8.59 (br s, 1H), 8.19 (d, J = 9.2 Hz, 2H), 7.78 (br d,
J = 26.1 Hz, 1H), 7.14 (d, J = 9.3 Hz, 2H), 6.93 (d, J = 4.9 Hz, 1H), 5.06 (d,
J = 3.9 Hz, 1H), 4.07 (ddd, J = 16.2, 10.0, 5.2 Hz, 2H), 3.89 (d, J = 3.9 Hz, 1H),
3.40–3.35 (m, 2H), 2.79–2.57 (m, 4H), 1.89 (br s, 1H). 13C NMR (151 MHz,
CDCl3) d 163.7, 162.7, 160.6 156.6 (dd, J = 36.2, 36.8 Hz), 141.9, 126.1, 120.6
(dd, J = 550.4, 274.9 Hz), 114.7, 105.9 (d, J = 2.3 Hz), 71.2, 68.4, 51.4, 48.9, 41.4;
LRMS (ESI+) m/z 402 (M+, 100%); HRMS (ESI) calcd for C16H18F3N5O4 (M+H)
402.1384, found 402.1380.
33. Usifoh C, Olugbade T, Onawumi G, Oluwadiya J.
J Heterocycl Chem.
1989;26:1069–1071.
sulphonamido-N-4-acetamide (1):
(4,6-Bis(20,40,3.4
tetramethoxyphenyl))pyrimidine-2-
solution of 4-amino-N-(4-(2,4-
A
dimethoxyphenyl)-6-(3,4-dimethoxyphenyl)pyrimidin-2-yl)benzenesulfonamide
9 (0.082 g, 0.157 mmol) in freshly distilled acetic anhydride (40 mL) was stirred at
50 °C for 16 h. The reaction mixture was then cooled to room temperature and
saturated NaHCO3 (40 mL) added dropwise followed by sonication for 30 min.
The resulting suspension was then filtered and washed with water (50 mL). The
precipitate was recrystallised from chloroform with drop wise addition of ethyl
acetate, to afford a yellow solid (0.058 g, 71%). 214–216 °C. 1H NMR (400 MHz,
DMSO-d6) d 11.56 (br s, 1H), 10.27 (s, 1H), 7.94 (d, J = 8.7 Hz, 2H), 7.79 (d,
J = 9.3 Hz, 1H), 7.71 (d, J = 8.8 Hz, 2H), 7.64–7.62 (m, 2H), 7.10 (d, J = 8.6 Hz, 1H),
6.71–6.68 (m, 2H), 3.89–3.84 (m, 12 H), 2.04 (s, 3H). 13C NMR (151 MHz, DMSO-
d6) d 172.0, 169.0, 163.6, 162.7, 159.4, 157.1, 151.4, 148.9, 143.0, 134.6, 131.8,
128.8, 128.5, 120.3, 118.2, 117.7, 111.7, 110.0 (2C), 105.9, 98.7, 55.9, 55.6, 55.6,
55.5, 24.1; LCMS (APCI+) m/z: 565 (M+H, 100%); HRMS (ESI) calcd for C28H28N4O7S
(M+H) 565.1751, found 565.1747.
38. ELISA based Screening: Purified full-length B. subtilis NusB was diluted to
250 nM in phosphate buffered saline (PBS) buffer and 100 lL of the solution
was added into NUNC MaxisorpTM microtitre plate wells. Following overnight
incubation with the NusB solution at 4 °C the wells were washed 3 times with
300 lL of PBS buffer and blocked with 300 lL of 1% (w/v) bovine serum
albumin (BSA) dissolved in PBS buffer at room temperature. After blocking for
2 h, plates were washed three times with wash buffer (PBS, 0.05% (v/v) Tween-
20). The appropriate inhibitor (Table 1) and 100 lL of affinity purified
glutathione-S-transferase (GST) tagged NusE at 200 nM were incubated at
37 °C for 15 min then were added to each well and incubated for 1 h at room
temperature. Unbound NusE was removed by washing each well 3 times in
34. Wasfy AAF, Aly AA. Folia Microbiol. 2003;48:51–55.
35. McCluskey A, Daniel JA, Hadzic G, et al. Traffic. 2013;14:1272–1289. (E)-3-(4-
hydroxyphenyl)-N0-(3,4,5-trimethoxybenzylidene)propanehydrazide (2): A solution
of 3,4,5-trimethoxybenzaldehyde 11 (0.06 g, 0.31 mmol) and 3-(4-
hydroxyphenyl)propane hydrazide (12) (0.06 g, 0.31 mmol) in ethanol (5 mL)
was heated at 60 °C 18 h. The resulting reaction mixture was then adsorbed to
silica and subject to column flash silica chromatography (4.2% CH3OH, in CH2Cl2)
to afford a white solid (0.097 g, 86%); mp 211–213 °C. 1H NMR (400 MHz,
CD3OD) d 7.93 (s, 1H), 7.16–7.01 (m, 4H), 6.96 (s, 1H), 6.70 (d, J = 7.9 Hz, 2H),
3.88 (s, 6H), 3.80 (s, 3H), 2.92 (t, J = 6.9 Hz, 2H), 2.55 (t, J = 7.5 Hz, 2H). 13C NMR
(101 MHz, CD3OD) d (major rotamer) 171.9, 156.7, 154.63, 148.9, 141.0, 132.6,
131.0, 130.28, 116.2, 106.0, 61.16, 56.7, 37.8, 31.8. 13C NMR (101 MHz, CD3OD) d
(minor rotamer) 177.1, 156.6, 154.68, 145.2, 140.8, 133.1, 131.39, 131.20, 116.1,
105.2, 61.18, 56.6, 36.0, 31.4. *1:0.3 ratio of major and minor rotamers. LRMS
(ESI+) m/z 359 (M+H, 100%); HRMS (ESI) calcd for C19H22N2O5 (M+Na) 381.1421,
found 381.1445.
300 lL of wash buffer. Rabbit anti-GST primary antibody (100 lL, 1:2000 in
PBS) was added to each well and incubated for 1 h at room temperature. After
washing, goat-anti-rabbit HRP secondary antibody (1:2000 in PBS) was added
to each well and incubated for 1 h at room temperature then washed 3 times in
300 lL of wash buffer. Visualisation of PPI was achieved by addition of 100 lL
TMB (3,30,5,50-tetramethylbenzidine liquid substrate system for ELISA, Sigma-
Aldrich) to each well. The plate was incubated in a plate reader (FLUOstar
Optima) at 37 °C with 200 rpm shaking for 6 min. The optical density of each
well was taken at 600 nm.
39. Baell JB, Holloway GA. J Med Chem. 2010;53:2719–2740.
40. Growth inhibition assay: The compounds were dissolved to 50 mM in DMSO and
serially diluted in 100
96-well NUNC MicrowellTM plate. E. coli DH5
37 °C in 5 mL LB with shaking until the optical density reached 0.6–0.7 AU, and
L of the culture was added to each well. The plate was incubated in the plate
l
L of Luria broth (LB) to a concentration of 200
lM in a
a
and B. subtilis were grown at
5
l
36. N,N0-[1,4-Butanediylbis(oxy-4,1-phenylene)]bis(N-ethyl)urea (3): To a solution of
1,4-bis(4-aminophenol)butane 16 (0.20 g, 0.76 mmol) in THF (10 mL) was
added ethyl isocyanate (0.13 mL, 1.62 mmol). The reaction mixture was then
heated at reflux overnight. The resulting reaction mixture was then cooled to
reader (FLUOstar Optima) at 37 °C with 200 rpm shaking. The optical density of
the culture was taken every 10 min using LB as the blank for 16 h at 600 nm.
The samples were tested in triplicate and the growth pattern of each sample
was compared to cells exposed to equal amounts of DMSO.