Structure–activity relationship studies of c-di-AMP synthase inhibitor, bromophenol-thiohydantoin

Article abstract of DOI:10.1016/j.tet.2015.10.073

C-di-AMP, a bacterial second messenger, regulates various processes in Gram-positive bacteria and mycobacteria. Small molecule inhibitors of c-di-AMP metabolic enzymes could affect bacterial growth and viability. A medium throughput screening identified b

Full text of DOI:10.1016/j.tet.2015.10.073

Accepted Manuscript
Structure-activity relationship studies of c-di-AMP synthase inhibitor, bromophenol-
thiohydantoin
Yue Zheng, Jie Zhou, Stefan M. Cooper, Jr., Clement Opoku-Temeng, Amanda
Moreira De Brito, Herman O. Sintim
PII:
S0040-4020(15)30173-3
10.1016/j.tet.2015.10.073
TET 27245
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 13 August 2015
Revised Date: 25 October 2015
Accepted Date: 26 October 2015
Please cite this article as: Zheng Y, Zhou J, Cooper Jr. SM, Opoku-Temeng C, De Brito AM, Sintim
HO, Structure-activity relationship studies of c-di-AMP synthase inhibitor, bromophenol-thiohydantoin,
Tetrahedron (2015), doi: 10.1016/j.tet.2015.10.073.
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Tetrahedron
journal homepage: www.elsevier.com
Structure-activity relationship studies of c-di-AMP synthase inhibitor, bromophenol-
thiohydantoin
Yue Zheng, Jie Zhou, Stefan M. Cooper Jr., Clement Opoku-Temeng, Amanda Moreira De Brito and
Herman O. Sintim
Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
C-di-AMP, a bacterial second messenger, regulates various processes in Gram-positive bacteria
and mycobacteria. Small molecule inhibitors of c-di-AMP metabolic enzymes could affect
bacterial growth and viability. A medium throughput screening identified bromophenol-
thiohydantoin (BTH) as the first inhibitor of c-di-AMP synthase, DisA. Herein, we performed
SAR studies of bromophenol-thiohydantoin to identify the salient features on BTH that are
important for DisA inhibition. Seemingly minor substitution changes (for example aromatic
bromo to chloro substitutions) resulted in dramatic changes in ligand potency. Bromophenol TH
is specific for c-di-AMP synthase and did not inhibit RocR (c-di-GMP PDE), YybT (c-di-AMP
PDE) or WspR (c-di-GMP synthase).
Available online
Keywords:
c-di-AMP
DisA
inhibitor
bromophenol-TH
SAR
2009 Elsevier Ltd. All rights reserved
.
———
Corresponding author. Tel.: +1-301-405-0633; fax: +1-301-314-9121; e-mail: hsintim@umd.edu

2
Tetrahedron
ACCEPTED MANUSCRIPT
1. Introduction
S
HN
NH
O
Multidrug-resistant bacteria continue to cause a threat to
Br
public health, despite decades of antibiotics development.1 The
US CDC in a report, emphasized the continuous need for new
antibiotics to keep up with the ever evolving antibacterial
resistance.2 Efforts have been put into the discovery of new types
of antibiotic targets. Enzymes that make or degrade bacterial
cyclic dinucleotide (cdn) second messengers or proteins/RNA
receptors of cdn have emerged as new antibacterial targets,
because cyclic dinucleotides regulate various cellular processes
in different bacterial species, including virulence, biofilms and
cell wall synthesis.3 Since its discovery in 2008,4 c-di-AMP has
been found in many Gram-positive bacteria and mycobacteria,
including human pathogens S. aureus,5 M. tuberculosis,6 L.
monocytogenes,7 and other bacteria.8 C-di-AMP is essential for
bacterial survival.7,9,10 In some bacteria, c-di-AMP has been
shown to regulate DNA integrity scanning,4 potassium ion
transport,11 bacterial cell wall homeostasis,12 fatty acid
synthesis13 and host type I interferon response induction14.
Attempts to delete c-di-AMP synthase, diadenylate cyclase
(DAC), from the genome of important human pathogens such as
S. aureus have been unsuccessful,15 implying that inhibitors of c-
di-AMP synthase could have antibacterial properties.
OH
Br
BTH
S
S
S
S
S
S
S
HN
HN
HN
HN
HN
HN
HN
NH
NH
NH
Br
NH
NH
NH
NH
O
O
O
O
O
O
O
H
F
Cl
Br
Cl
F
OH
OH
OH
OH
OH
OH
OH
Br
Br
Br
F
4
H
5
I
Cl
7
1
2
3
6
S
S
S
S
HN
HN
HN
HN
NH
NH
NH
NH
O
O
O
O
H
F
Cl
I
OH
OH
OH
OH
H
8
F
9
Cl
10
I
11
S
S
S
HN
HN
HN
NH
NH
NH
O
O
O
Br
Br
Br
OCH3
NH2
H
Br
Br
Br
12
13
14
S
O
O
H3C
S
H3C
S
HN
HN
S
N
N
S
NH
NH
NH
N
CH3
O
O
O
O
O
Br
Br
Br
Br
Br
OH
OH
OH
OH
OH
Br
15
Br
Br
Br
18
Br
19
Motivated by the potential utility of DAC inhibitors as
important probes to study c-di-AMP signaling in bacteria or as
new generation antibiotics we embarked on the development of a
high throughput assay, using coralyne, to discover such
inhibitors.16 Using the coralyne assay, we identified the first c-di-
16
17
Figure 1. Structures of BTH and analogs synthesized for this
study. Ninteen BTH analogs are grouped into four categories,
accoding to their substitution types.
AMP
synthase
inhibitor,
5-(3,
5-dibromo-2-
hydroxylbenzylidene)-2-thioxoimidazolidin-4-one (referred to as
bromophenol thiohydantoin or bromophenol-TH or BTH, see
Figure 1 for structure).17 BTH contains a halogen (Br), phenolic
and 2-thiohydantoin (or 2-thioxoimidazolidin-4-one) moieties.
To guide the development of BTH-based/inspired DAC
inhibitors, we initiated a structure–activity relationship (SAR)
study to tease out the salient features of BTH that are important
for DisA inhibition. Herein, we present the synthesis of 19 BTH
analogs, and investigate the importance of the various
functionalities found on BTH for DAC inhibition.
General synthesis procedure C: A mixture of aldehyde (0.5
mmol, 1 equiv. 20), compound 21 (1.1 equiv.), and NH4OAc (1.5
equiv.) in toluene (4 mL) was stirred at 80-90 C (12 h) until no
starting material remained, assessed by TLC (SiO2, hexane/ethyl
acetate, 1:1, v/v). The reaction mixture was cooled down to room
temperature. The resulting precipitate was filtered and washed
successively with water and Et2O (10 mL).
o
5-(3-bromo-2-hydroxybenzylidene)-2-thioxoimidazolidin-4-
one (compound 1)
2. Experimental section
Following synthesis procedure A, compound 1 was obtained
as brown solid. Compound 1 (38 mg, 50% yield) H NMR (500
1
2.1 Synthesis of BTH analogs
MHz, MeOD) δ 7.52 (dd, J = 7.9, 1.1 Hz, 1H), 7.46 (d, J = 7.6
Hz, 1H), 6.87 (t, J = 7.9 Hz, 1H), 6.75 (s, 1H). 13C NMR (126
MHz, MeOD) δ 180.04, 167.60, 153.54, 135.17, 131.06, 130.24,
124.22, 122.78, 112.74, 108.71. HRMS (ESI-) m/z calcd for
C10H6BrN2O2S [M-H]- 296.9333, found 296.9350.
Three different synthetic protocols were explored for the
synthesis of the analogs and the one that gave the best yield was
chosen for making adequate amounts for characterization and
biological evaluation.
5-(3-bromo-5-fluoro-2-hydroxybenzylidene)-2-
thioxoimidazolidin-4-one (compound 2)
General synthesis procedure A: A mixture of compound 20,
aldehyde (0.5 mmol, 1 equiv. 20), compound 21 (1.2 equiv.), and
NaOAc (3 equiv.) in glacial AcOH (4 mL) was stirred at reflux
(12 h) until no starting material remained, assessed by TLC
(SiO2, hexane/ethyl acetate, 1:1, v/v). The reaction mixture was
cooled down to room temperature, followed by the addition of
iced water. The resulting precipitate was filtered and washed
successively with water and Et2O (10 mL). The product was
subjected to column purification (hexane/ethyl acetate, 0ꢀ35%).
Following synthesis strategy A, compound 2 was obtained as
1
yellow solid. Compound 2 (68 mg, 29% yield) H NMR (500
MHz, MeOD) δ 7.34 (s, 1H), 7.18 (s, 1H), 6.60 (s, 1H). 13C NMR
(126 MHz, DMSO) δ 177.94, 167.02, 156.30, 154.39, 152.99,
129.99, 123.79, 121.28, 121.08, 116.87, 116.70, 113.55, 109.10.
HRMS (ESI-) m/z calcd for C10H5BrFN2O2S [M-H]- 314.9239,
found 314.9251.
General synthesis procedure B: A mixture of aldehyde (0.5
mmol, 1 equiv. 20), compound 21 (1.2 equiv.), and few drops of
piperidine in EtOH (4 mL) was stirred at 80-90 C (4-12 h) until
5-(3-bromo-5-chloro-2-hydroxybenzylidene)-2-
thioxoimidazolidin-4-one (compound 3)
o
no starting material remained, assessed by TLC (SiO2,
hexane/ethyl acetate, 1:1, v/v). The reaction mixture was cooled
down to room temperature. The resulting precipitate was filtered
and washed successively with water and Et2O (10 mL).
Following synthesis strategy A, compound 3 was obtained as
brown solid. Compound 3 (28 mg, 12% yield) H NMR (600
1
MHz, MeOD) δ 7.55 (d, J = 2.3 Hz, 1H), 7.48 (d, J = 2.4 Hz,
1H), 6.64 (s, 1H). 13C NMR (151 MHz, MeOD) δ 178.09,

3
165.50, 151.50, 131.99, 129.08, 128.37, 124.07, 123.32, 111.48,
105.34. HRMS (ESI-) m/z calcd for C10H5BrClN2O2S [M-H]-
330.8944, found 330.8963
128.93 (s), 122.97 (dd, J = 9.4, 4.1 Hz), 111.46 (s), 111.28 (d, J
ACCEPTED MANUSCRIPT
= 3.2 Hz), 106.64 (s), 104.77 (s), 104.56 (d, J = 3.8 Hz), 104.36
(s). HRMS (ESI-) m/z calcd for C10H5F2N2O2S [M-H]- 255.0040,
found 255.0049.
5-(5-bromo-3-fluoro-2-hydroxybenzylidene)-2-
thioxoimidazolidin-4-one (compound 4)
5-(3,5-dichloro-2-hydroxybenzylidene)-2-thioxoimidazolidin-
4-one (compound 10)
Following synthesis strategy B, compound 4 was obtained as
1
yellow solid. Compound 4 (132 mg, 57% yield) H NMR (400
Following synthesis strategy A, compound 10 was obtained as
1
MHz, DMSO) δ 7.36 (s, 1H), 7.30 (d, J = 10.3 Hz, 1H), 6.33 (s,
1H). 13C NMR (151 MHz, DMSO) δ 175.39, 166.19, 154.37,
152.77, 149.94, 130.98, 129.69, 125.29, 125.26, 118.88, 118.73,
108.95, 104.01. HRMS (ESI-) m/z calcd for C10H5BrFN2O2S [M-
H]- 314.9239, found 314.9217.
brown solid. Compound 10 (58 mg, 78% yield) H NMR (500
MHz, MeOD) δ 7.19 (s, 1H), 7.01 (s, 1H), 6.30 (s, 1H). 13C NMR
(126 MHz, MeOD) δ 180.47, 168.16, 163.51, 133.26, 131.80,
128.79, 128.12, 125.62, 117.68, 116.00. HRMS (ESI-) m/z calcd
for C10H5Cl2N2O2S [M-H]- 286.9449, found 286.9476.
5-(5-bromo-2-hydroxybenzylidene)-2-thioxoimidazolidin-4-
one (compound 5)
5-(2-hydroxy-3,5-diiodobenzylidene)-2-thioxoimidazolidin-4-
one (compound 11)
Following synthesis strategy B, compound 5 was obtained as
yellow solid. Compound 5 (156 mg, 67% yield) H NMR (600
Following synthesis strategy B, compound 11 was obtained as
yellow solid. Compound 11 (100 mg, 50% yield) H NMR (600
1
1
MHz, DMSO) δ 7.39 (d, J = 2.5 Hz, 1H), 7.16 (dd, J = 8.7, 2.6
Hz, 1H), 6.57 (d, J = 8.7 Hz, 1H), 6.01 (s, 1H). 13C NMR (151
MHz, DMSO) δ 176.18, 168.36, 158.56, 137.42, 134.49, 132.17,
124.56, 120.73, 108.95, 107.74. HRMS (ESI-) m/z calcd for
C10H6BrN2O2S [M-H]- 296.9333, found 296.9356.
MHz, DMSO) δ 7.69 (s, 1H), 7.36 (d, J = 1.9 Hz, 1H), 6.05 (s,
1H). 13C NMR (151 MHz, DMSO) δ 172.31, 166.00, 164.21,
145.84, 142.32, 129.76, 123.86, 112.94, 97.38, 71.81. HRMS
(ESI-) m/z calcd for C10H5I2N2O2S [M-H]- 470.8161, found
470.8165.
5-(5-chloro-2-hydroxy-3-iodobenzylidene)-2-
thioxoimidazolidin-4-one (compound 6)
5-(3,5-dibromo-2-methoxybenzylidene)-2-thioxoimidazolidin-
4-one (compound 12)
Following synthesis strategy B, compound 6 was obtained as
Following synthesis strategy C, compound 12 was obtained as
brown solid, a mixture of E/Z isomers. Compound 12 (25 mg,
12% yield) H NMR (400 MHz, DMSO/D2O) δ 8.69 (s, 1H),
8.57 (s, 0.26H), 7.85 (s, 0.3H), 7.68 (d, J = 1.9 Hz, 1H), 6.66 (s,
0.28H), 6.16 (s, 1H), 4.08 (s, 1H), 3.73 (s, 3H).13C NMR (151
MHz, DMSO/D2O) δ 183.85, 183.38, 182.96, 174.48, 172.03,
170.35, 153.68, 133.00, 132.45, 131.93, 117.44, 116.94, 98.03,
50.25. HRMS (ESI-) m/z calcd for C11H7Br2N2O2S [M-H]-
388.8595, found 388.8583.
1
yellow solid. Compound 6 (120 mg, 56% yield) H NMR (600
MHz, DMSO) δ 7.53 (s, 1H), 7.19 (s, 1H), 6.09 (s, 1H). 13C
NMR (151 MHz, DMSO) δ 172.79, 166.19, 162.94, 138.04,
133.27, 130.73, 121.58, 115.99, 112.63, 95.29. HRMS (ESI-) m/z
calcd for C10H5ClIN2O2S [M-H]- 378.8805, found 378.8813.
1
5-(3-chloro-5-fluoro-2-hydroxybenzylidene)-2-
thioxoimidazolidin-4-one (compound 7)
Following synthesis strategy B, compound 7 was obtained as
yellow solid, a mixture of E/Z isomers. Compound 7 (153 mg,
61% yield) 1H NMR (600 MHz, DMSO) δ 8.19 (dd, J = 10.3, 3.1
Hz, 0.1H), 7.38 (dd, J = 8.0, 3.1 Hz, 0.1H), 7.20 (dd, J = 8.0, 3.2
Hz, 1H), 7.09 (dd, J = 9.9, 3.1 Hz, 1H), 6.86 (s, 0.1H), 6.17 (s,
1H). 13C NMR (151 MHz, DMSO) δ 176.07, 175.23, 166.85,
163.54, 154.78, 154.13, 153.21, 152.64, 151.10, 148.38, 133.48,
130.94, 123.47, 123.41, 123.17, 123.09, 123.06, 123.00, 121.58,
121.50, 117.57, 117.35, 117.18, 116.89, 116.75, 115.09, 114.93,
111.49, 110.00. HRMS (ESI-) m/z calcd for C10H5ClFN2O2S [M-
H]- 270.9744, found 270.9723.
5-(2-amino-3,5-dibromobenzylidene)-2-thioxoimidazolidin-4-
one (compound 13)
Following synthesis strategy B, compound 13 was obtained as
yellow solid, a mixture of E/Z isomers. Compound 13 (86 mg,
1
30% yield) H NMR (500 MHz, DMSO/D2O) δ 9.39 (s, 0.3H),
7.91 (s, 0.3H), 7.89 (s, 0.3H), 7.58 (d, J = 2.0 Hz, 1H), 7.41 (s,
1H), 6.43 (s, 1H). 13C NMR (126 MHz, DMSO/D2O) δ 181.92,
179.82, 166.35, 159.34, 144.82, 135.60, 134.09, 132.50, 132.38,
131.87, 130.67, 130.26, 123.89, 121.09, 119.50, 115.56, 110.34,
109.95, 108.15, 107.78. HRMS (ESI-) m/z calcd for
C10H6Br2N3OS [M-H]- 373.8598, found 373.8589.
5-(2-hydroxybenzylidene)-2-thioxoimidazolidin-4-one
(compound 8)
5-(3,5-dibromobenzylidene)-2-thioxoimidazolidin-4-one
(compound 14)
Following synthesis strategy A, compound 8 was obtained as
1
brown solid. Compound 8 (213 mg, 59% yield) H NMR (500
Following synthesis strategy B, compound 14 was obtained as
1
MHz, MeOD) δ 7.36 (d, J = 7.5 Hz, 1H), 7.21 (t, J = 7.4 Hz, 1H),
6.99 – 6.73 (m, 2H), 6.64 (s, 1H). 13C NMR (126 MHz, MeOD) δ
180.41, 168.49, 158.36, 133.73, 132.72, 129.55, 121.82, 120.80,
118.03, 112.52. HRMS (ESI-) m/z calcd for C10H5N2O2S [M-H]-
219.0228, found 219.0240.
yellow solid. Compound 14 (45 mg, 20% yield) H NMR (600
MHz, DMSO) δ 8.22 (s, 2H), 7.58 (s, 1H), 5.95 (s, 1H). 13C NMR
(151 MHz, DMSO) δ 183.21, 183.09, 170.83, 140.49, 130.56,
130.50, 122.27, 103.54. HRMS (ESI-) m/z calcd for
C10H5Br2N2OS [M-H]- 358.8489 found 358.8506.
5-(3,5-difluoro-2-hydroxybenzylidene)-2-thioxoimidazolidin-
4-one (compound 9)
5-(3,5-dibromo-2-hydroxybenzylidene)-2-thioxothiazolidin-4-
one (compound 15)
Following synthesis strategy A, compound 9 was obtained as
brown solid. Compound 9 (72 mg, 89% yield) H NMR (500
Following synthesis strategy A, compound 15 was obtained as
brown solid. Compound 15 (28 mg, 13% yield) H NMR (500
1
1
MHz, MeOD) δ 7.05 (d, J = 9.2 Hz, 1H), 6.99 (ddd, J = 11.0, 8.3,
2.9 Hz, 1H), 6.63 (s, 1H). 13C NMR (126 MHz, MeOD) δ 177.80,
166.09, 155.31 (d, J = 11.8 Hz), 153.42 (d, J = 11.7 Hz), 152.91
(d, J = 12.8 Hz), 151.00 (d, J = 12.8 Hz), 141.64 (d, J = 13.7 Hz),
MHz, MeOD) δ 7.83 (s, 1H), 7.75 (d, J = 2.2 Hz, 1H), 7.42 (d, J
= 2.2 Hz, 1H). 13C NMR (126 MHz, MeOD) δ 195.71, 170.06,
153.56, 137.18, 131.22, 128.96, 125.82, 125.50, 113.12, 112.51.

4
Tetrahedron
HRMS (ESI-) m/z calcd for C10H4Br2NO2S2 [M-H]- 391.8050,
BTH/analogs and 1 ꢀM DisA were mixed in reaction buffer,
ACCEPTED MANUSCRIPT
found 391.8072.
which contains 40 mM Tris-HCl, pH 7.5, 100 mM NaCl and 10
mM MgCl2. Fluorescence measurements were performed on a
Molecular Devices SpectraMax M5e microplate reader (λex=420
nm and λem=475 nm). Reactions were monitored for 30 min with
2 min interval.
5-(3,5-dibromo-2-hydroxybenzylidene)imidazolidine-2,4-
dione (compound 16)
Following synthesis strategy A, compound 16 was obtained as
1
brown solid. Compound 16 (52 mg, 25% yield) H NMR (400
2.4 IC50 measurements of BTH, compound 5 and compound 6
MHz, MeOD) δ 7.61 (d, J = 2.3 Hz, 1H), 7.50 (d, J = 2.3 Hz,
1H), 6.64 (s, 1H). 13C NMR (151 MHz, MeOD) δ 167.28, 157.11,
154.27, 136.19, 132.80, 130.84, 126.27, 114.05, 112.25, 105.95.
HRMS (ESI-) m/z calcd for C10H5Br2N2O3 [M-H]- 358.8667,
found 358.8685.
300 ꢀM ATP, 33 nM 32P-ATP and various concentrations of
BTH/analogs were mixed in reaction buffer. Reaction was
initiated by addition of 1 ꢀM DisA and performed at 30 °C for 1
hour. Aliquots of the reaction mixtures were spotted on a TLC
plate (EMD Millipore TLC Cellulose). TLC running buffer was
saturated (NH4)2SO4 : 1.5 M KH2PO4 = 1:1.5.
4-(3,5-dibromo-2-hydroxybenzylidene)thiazolidine-2,5-dione
(compound 17)
2.5 Assay to determine if BTH is a covalent inhibitor
Following synthesis strategy C, compound 17 was obtained as
brown solid. Compound 17 (146 mg, 67 % yield) H NMR (600
1
10 ꢀM DisA was incubated with 100 ꢀM BTH or DMSO on
ice for 90 min. BTH/DMSO was removed by filtration. DisA was
washed by reaction buffer for three times. DisA was then
incubated with 300 ꢀM ATP, 10 ꢀM coralyne and 3 mM KI.
MHz, MeOD) δ 8.06 (s, 1H), 7.56 (d, J = 2.4 Hz, 1H), 7.40 (d, J
= 2.4 Hz, 1H). 13C NMR (151 MHz, DMSO) δ 173.64, 172.65,
157.59, 134.04, 128.65, 125.05, 124.81, 124.28, 113.57, 105.24.
HRMS (ESI-) m/z calcd for C10H4Br2NO3S [M-H]- 375.8279,
found 375.8282.
Reaction was monitored by fluorescence emission on
a
Molecular Devices SpectraMax M5e microplate reader (λex=420
nm and λem=475 nm). The reaction was monitored for 30 min
with 5 min interval.
5-(3,5-dibromo-2-hydroxybenzylidene)-3-methyl-2-
thioxoimidazolidin-4-one (compound 18)
2.6 Effect of BTH on YybT, WspR D70E and RocR activity
Following synthesis strategy B, compound 18 was obtained as
yellow solid. Compound 18 (201 mg, 89 % yield) H NMR (600
1
1.5 ꢀM YybT reacted with 50 ꢀM c-di-AMP and 16 nM 32P-c-
di-AMP in the presence or absence of 100 ꢀM BTH in reaction
buffer (100 mM Tris-HCl, pH 8.3, 20 mM KCl, 0.5 mM MnCl2
and 1 mM DTT) at 37 °C. The reaction was stopped after 30 min.
1 ꢀM WspR D70E reacted with 300 ꢀM GTP and 33 nM 32P-
GTP in the presence or absence of 100 ꢀM BTH in reaction
buffer (10 mM Tris-HCl, pH 8.0, 100 mM NaCl and 5 mM
MgCl2) at 37 °C. Reaction was stopped after 2 hours. 1.2 ꢀM
RocR reacted with 50 ꢀM c-di-GMP and 16 nM 32P-c-di-GMP in
reaction buffer (100 mM Tris-HCl, pH 8.0, 20 mM KCl and 25
mM MgCl2) at 37 °C. Reaction was stopped after 30 min. All the
reactions were monitored by TLC.
MHz, DMSO) δ 7.46 (d, J = 2.6 Hz, 1H), 7.29 (d, J = 2.6 Hz,
1H), 6.25 (s, 1H), 3.10 (s, 3H). 13C NMR (151 MHz, DMSO) δ
173.41, 164.62, 160.61, 135.29, 135.12, 129.68, 124.54, 116.92,
113.65, 102.85, 27.00. HRMS (ESI-) m/z calcd for
C11H7Br2N2O2S [M-H]- 388.8595, found 388.8571.
5-(3,5-dibromo-2-hydroxybenzylidene)-1,3-dimethyl-2-
thioxoimidazolidin-4-one (compound 19)
Following synthesis strategy B, compound 19 was obtained as
1
yellow solid. Compound 19 (156 mg, 67% yield) H NMR (600
MHz, DMSO) δ 8.63 (d, J = 2.5 Hz, 1H), 7.44 (d, J = 2.6 Hz,
1H), 7.21 (s, 1H), 3.50 (s, 3H), 3.20 (s, 3H). 13C NMR (151 MHz,
DMSO) δ 173.99, 163.12, 161.15, 135.74, 131.56, 125.33,
122.68, 119.73, 116.45, 100.24, 30.35, 27.82.HRMS (ESI-) m/z
calcd for C12H9Br2N2O2S [M-H]- 402.8751, found 402.8790.
3
Results and Discussion
BTH, is easily prepared via the condensation of 3, 5-
dibromosalicylic
aldehyde
and
2-thiohydantoin.18-20
Conveniently, this ease of preparation allows for facile
preparation of analogs via condensation reactions with various
commercially available halo-substituted salicylic aldehydes and
hydantoins, thiohydantoins or rhodanines. (Scheme 1). Electronic
and steric derivations are possible by varying the halogens
present on the aromatic ring.
2.2 Protein purification
BL21(DE3) cells with protein expression plasmids were
cultured in LB medium with 100 ꢀg/ml ampicillin or 50 ꢀg/ml
kanamycin at 37 °C. Expression was induced by addition of 1
mM IPTG. After induction at 30 °C for 6 hours (WspR D70E and
YybT) or at 16 °C overnight (DisA and RocR), cells were
harvested by centrifugation at 4,000 rpm for 15 min. Cells were
resuspended in lysis buffer (10 mM Tris-HCl, pH 8.0, 100 mM
NaCl for WspR D70E, RocR and YybT, and 50 mM sodium
phosphate buffer, pH 8.0, 300 mM NaCl for DisA) and lysed by
sonication. Proteins were purified by GE Hitrap Nickel columns
and dialyzed into lysis buffer.
Four types of BTH analogs (A, B, C, D, see Figure 1) were
prepared. In type A, we synthesized analogs containing mixed
halogens whereas type B consisted of only one type of halogens.
In type C, we investigated the role of the phenolic group in BTH
by replacing the OH with hydrogen, amino or methoxy groups.
Type D explored how subtle changes to the heterocyclic ring of
BTH would affect DisA inhibition.21-23
2.3 The coralyne assay
3.1 Inhibition of DisA by BTH analogs
BTH and analogs were stored as 100 mM stock solutions in
DMSO. 300 ꢀM ATP, 10 ꢀM coralyne, 3 mM KI, 50 ꢀM

5
ACCEPTED MANUSCRIPT
Scheme 1. Synthetic strategy for preparing BTH and analogs. Conditions for method A, B and C are listed above the
arrow. The method that gave the best yield was chosen for each compound.
The coralyne assay16 (Supplementary Material, Figure S1) was
performed to identify the DisA inhibition effects of BTH analogs.
In the absence of c-di-AMP, iodide quenches corlayne
fluorescence. As DisA synthesizes c-di-AMP, the dinucleotide
forms a complex with coralyne and protects coralyne from
quenching effects. Coralyne fluorescence emission intensity
correlates with c-di-AMP concentration. In the presence of
inhibitors, the concentration of c-di-AMP would be lower and
hence the fluorescence of coralyne would be reduced.16 The
coralyne assay indicated that BTH was the strongest inhibitor,
followed by compound 15 in group D and compound 3 in group
A. Other BTH analogs did not show significant inhibition of
DisA. IC50 of BTH (6.7 ± 0.6 × 10-5 M), compound 15 (12.4 ±
1.0 × 10-5 M) and compound 3 (17.9 ± 1.7 × 10-5 M) were
obtained using radiolabeled 32P-ATP (Figure 2). In group A,
BTH, compound 1, compound 2 and compound 3 have 3’-bromo
and their inhibition effects increase as 5’-halogen size increases.
It appears that the bromo substitution at the 3-position is
important as deleting this substitution (compound 5) resulted in
loss of inhibition. Compound 6 or 7, which replaced the dibromo
with mixed halogens (Cl, F or I) were also inactive, highlighting
the need for bromo substitution.
the thiohydantoin group also eliminated inhibition by the BTH
analog (compare compound 18 with BTH), Supplementary
Material, Figure S1. compound 19, the dimethyl thiohydantoin
analog was also not active.
3.2 BTH is a non-covalent inhibitor
BTH is reported to be a non-competitive inhibitor17, although
the binding site for inhibitors within DisA is currently unknown.
BTH contains an enone moiety and hence could act via covalent
inhibition. To ascertain if covalent inhibition was operative, DisA
was incubated with high concentration of BTH and then unbound
BTH was removed by filtration. The remaining DisA was then
washed three times with reaction buffer and then assayed for
activity. The BTH-treated DisA retained enzymatic activity
(similar to DMSO-treated DisA, see Supplementary Material,
Figure S2), revealing that BTH and analogs are not covalent
inhibitors.
3.3 BTH did not affect YybT, WspR D70E and RocR activity
We were curious to know the selectivity of BTH and analogs
towards cyclic dinucleotide metabolism enzymes. We therefore
tested BTH against a few cyclic dinucleotide-related enzymes,
namely c-di-AMP PDE YybT from B. subtilis24, c-di-GMP DGC
WspR D70E25 and c-di-GMP PDE RocR26 from P. aeruginosa.
Interestingly 100 ꢀM of BTH did not affect any of these enzymes
(Supplementary Material, Figure S3), implying that BTH is
indeed specific for c-di-AMP synthase, DisA.
The diiodinated analog compound 11 of group B showed more
inhibition than the non-halogenated analog compound 8,
difluorinated compound 9 and dichlorinated analog compound 10,
but it was still less active than BTH (see Supplementary Material,
Figure S1). The hydroxyl group in BTH was replaced with a
methoxy (compound 12), amino (compound 13) and hydrogen
(compound 14) to investigate if the phenolic moiety played a role
in inhibition. In all of the three cases, replacement of the phenolic
group with the aforementioned moieties completely diminished
any inhibition effects. In group D, substitution of the nitrogen
atom for sulfur atom at 1’ position (2-thioxoimidazolidin-4-one
to 2-thioxothiazolidin-4-one, compare compound 15 with BTH)
has minor impact on inhibition, whereas replacement of the
thiocarbonyl by carbonyl (2-thioxoimidazolidin-4-one to
imidazolidine-2,4-dione, compare compound 16 with BTH)
strongly reduced the inhibition effects. Substitution of the N-3 of
4
Conclusion.
Cyclic dinucleotides have emerged as interesting second
messengers in both prokaryotes and eukaryotes and there has
been an explosion of research activities to unravel the intricacies
of signaling regulated by these nucleotides. There is however a
paucity of small molecules that inhibit the metabolic enzymes of
cyclic dinucleotides and such molecules could have important
roles in elucidating cdn signaling and/or become next generation
antibacterial agents. In this paper, we have demonstrated that
bromophenol TH, the first reported inhibitor of c-di-AMP
synthase is sensitive to modifications and most changes to this
inhibitor abrogated inhibition. Interestingly, seemingly minor
changes (for example changing an aromatic bromo to a chloro or
iodo substituent) resulted in drastic inhibition profile,
highlighting that halogen substitution can be used to cause
dramatic changes in enzyme affinity for a ligand. Despite the
presence of an enone moiety, bromophenol TH is not a covalent
inhibitor of DisA. Future work will involve the search of other
heterocyclic groups, apart from hydantoin or rhodanines, which
could be appended to the bromophenol moiety with the ultimate
goal of discovering more potent inhibitors of DisA.
BTH
Compound 15
Compound 3
Acknowledgments
Figure 2. Enzymatic proficiency of DisA, 1 ꢀM, (converting 32P-
ATP into 32P-c-di-AMP) in the presence of increasing amounts of
inhibitors BTH, compound 15 and compound 3.

6
Tetrahedron
ACCEPTED MANUSCRIPT
15. Mehne, F. M.; Gunka, K.; Eilers, H.; Herzberg, C.; Kaever, V.; Stülke,
We sincerely thank Dr. Karl-Peter Hopfner and Dr. Angelika
J. J. Biol. Chem. 2013, 288, 2004-17.
Gründling for providing DisA plasmids and Dr. Zhaoxun Liang
for providing RocR and YybT plasmids. Funding for this work
was provided by the National Science Foundation
(CHE1307218), Camille Dreyfus Foundation and Kraybill
Biochemistry Fellowship (YZ).
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The coralyne assay to determine the inhibition profile of
analogs, determination of whether BTH is a covalent inhibitor
and the activity of BTH towards other cyclic dinucleotide-related
enzymes.