Derivatives of 8-hydroxyquinoline - Antibacterial agents that target intra- and extracellular Gram-negative pathogens

Article abstract of DOI:10.1016/j.bmcl.2012.03.096

Small molecule screening identified 5-nitro-7-((4-phenylpiperazine-1-yl-) methyl)quinolin-8-ol INP1750 as a putative inhibitor of type III secretion (T3S) in the Gram-negative pathogen Yersinia pseudotuberculosis. In this study we report structure-activit

Full text of DOI:10.1016/j.bmcl.2012.03.096

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3550–3553
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Derivatives of 8-hydroxyquinoline—antibacterial agents that target
intra- and extracellular Gram-negative pathogens
Per-Anders Enquist a,b, Åsa Gylfe b,c, Ulrik Hägglund d, Pia Lindström d, Henrik Norberg-Scherman d,
a,b,
Charlotta Sundin d, , Mikael Elofsson
⇑
⇑
a Laboratories for Chemical Biology Umeå, Department of Chemistry, Umeå University, SE90187 Umeå, Sweden
b Umeå Centre for Microbial Research and Laboratories for Molecular Infection Medicine Sweden, Umeå University, SE90187 Umeå, Sweden
c Clinical Microbiology, Umeå University, SE90187 Umeå, Sweden
d Creative Antibiotics, Tvistevägen 48, SE90719 Umeå, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
Small molecule screening identified 5-nitro-7-((4-phenylpiperazine-1-yl-)methyl)quinolin-8-ol INP1750
as a putative inhibitor of type III secretion (T3S) in the Gram-negative pathogen Yersinia pseudotubercu-
losis. In this study we report structure–activity relationships for inhibition of T3S and show that the most
potent compounds target both the extracellular bacterium Y. pseudotuberculosis and the intracellular
pathogen Chlamydia trachomatis in cell-based infection models.
Received 10 February 2012
Revised 9 March 2012
Accepted 10 March 2012
Available online 6 April 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Type III secretion
Antibacterial
Yersinia
Chlamydia
Screening
Resistance to antibiotics is one of the major challenges facing
treatment of bacterial infections, highlighting the need for novel
antibacterial compounds that are effective towards resistant
strains. The compounds should have novel modes of action and
structural scaffolds different from those of current antibiotics. Such
compounds should show antibacterial activity against resistant
strains and a responsible use could possibly reduce the frequency
of resistance to the new drugs.
Many clinically relevant Gram-negative bacteria utilize T3S to
inject toxins into the cytosol of eukaryotic cells and thereby create
an environment that allows the bacteria to grow and establish an
infection.1 We have previously used a bacterial reporter-gene assay
in Yersinia pseudotuberculosis to screen a 9400 compound library
for putative inhibitors of bacterial type III secretion (T3S).2 Three
classes of compounds have been pursued3–9 and inhibitors active
against multiple species including Yersinia, Salmonella, Chlamydia,
Escherichia coli, and Shigella were identified.3,10–16 Compounds
have been tested in vivo and the results indicate that T3S inhibitors
have a potential for therapy or prevention.14,17,18
new derivatives, pseudoceramine A–D, from a marine sponge
Pseudoceratina sp. as general antibiotics.19
Based on the experience from the first screening campaign and
post-screening activities we decided to screen an additional collec-
tion consisting of 17,500 synthetic small organic molecules with
the hope to find additional T3S inhibitors belonging to new
chemotypes. The 8-hydroxyquinoline derivative INP1750 (Scheme
1, Table 1) was identified as a single hit.20 INP1750 and analogs can
conveniently be assembled in a one-step synthesis according to
classic Mannich chemistry (Scheme 1).21
We decided to establish structure–activity relationships by
synthesis of compounds using commercially available 8-hydroxy-
NO2
NO2
N
N
OH
Formaldehyde
1
N
Pyridine, 50 °C,
N
The T3S reporter-gene assay has also been used in a large screen
to identify the known natural product spermatinamine and four
+
30 min, (95%)
OH
N
INP1750
HN
⇑
2
Corresponding authors. Tel.: +46 90 7869328 (M.E.), +46 90 136650 (C.S.).
E-mail addresses: charlotta.sundin@creativeantibiotics.com (C. Sundin), mikael.
elofsson@chem.umu.se (M. Elofsson).
Scheme 1. Synthesis of INP1750.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.096

P.-A. Enquist et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3550–3553
3551
Table 1
Effect of the synthesized compounds on the reporter-gene signal in Y. pseudotuberculosis
Substance
Reporter gene inhibitiona
Substance
Reporter gene inhibitiona
20 50
10
66
l
M
20
88
l
M
50
94
l
M
100
lM
10
l
M
l
M
l
M
100 lM
17b
56b
88b
94b
F
F
97
NO2
NO2
49b
73b
77b
79b
38b,c
66b,c
81b,c
83b,c
N
N
N
N
N
N
OH
OH
INP1750
INP1855
NA
NA
14
9
Cl
Cl
NAc
NAc
22c
74c
N
N
N
N
NA
NA
NAc
NA
NA
NA
NAc
NA
93
97
N
N
OH
OH
INP1813
INP1788
F
F
Cl
Br
N
N
N
N
NA
NA
NA
NA
78
89
N
O
N
OH
INP1869
INP1769
Br
N
N
N
N
NAc
NAc
NAc
NAc
58c
NA
95c
NA
N
O
N
OH
INP1860
INP1870
Cl
NO2
NO2
N
N
N
NAc
NAc
NAc
NAc
N
OH
OH
INP1853
INP1758
NO2
54
79
95
98
NO2
47b
64b
68b
69b
N
N
NA
NA
NA
NA
NA
25
NA
41
77
NA
81
85
N
N
OH
Cl
OH
INP1767
INP1759
Br
N
N
N
N
NA
NA
NA
NA
NA
48
12
N
N
N
N
OH
Cl
OH
INP1858
INP1765
NO2
N
N
N
OMe
8
OH
OH
Cl
INP1764
INP1772
NO2
O
N
N
N
OEt
N
NA
NA
NA
NA
37
80
89
98
NA
NA
76
93
N
N
N
OH
OH
Cl
INP1817
INP1766
N
N
OH
INP1768
NA = no activity.
a
Percent inhibition.
b
Percent inhibition of phosphatase activity from secreted YopH.
Experiment performed on the HCl salt.
c
quinolines, phenols, and cyclic secondary amines. In total 19
compounds22 were screened in the original T3S-linked reporter-
gene assay at four concentrations (Table 1).2,6 The three most potent
compounds, INP1750, INP1767, and INP185523 were then tested for
inhibition of bacterial growth and inhibition of phosphatase activity
originating from the secreted tyrosine phosphatase YopH as de-
scribed previously.6,7 The compounds showed a clear dose-response
effect in both the reporter-gene assay and the YopH assay (Table 1)

3552
P.-A. Enquist et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3550–3553
fected with wild-type Y. pseudotuberculosis. The T3S system trans-
locates toxins into the macrophage cytosol resulting in cell death
or reduced viability. The status of the J774 cells was monitored
using CalceinAM, which is converted to a green fluorescent mole-
cule in healthy cells. The bacteria cause a reduction of cell viability
and this effect was reversed by all three compounds (Fig. 2a). The
compounds show little effect when the macrophages are infected
with a non-virulent T3S-deficient mutant (Fig. 2b) and display
modest toxicity as observed for treated uninfected macrophages
(Fig. 2c).
We have previously shown that compounds active in
Y. pseudotuberculosis are active against multiple bacterial species
including Chlamydia. As a next step we evaluated INP1750,
INP1767, and INP1855 against Chlamydia trachomatis in an
ex vivo infection model as described previously.6,26 All three
compounds proved to be active also against C. trachomatis with
minimum inhibitory concentrations (MICs) of 3–25 lM (Table 2).
Cell viability was scored with rezazurin27,28 at concentrations
twice the MIC values. INP1750 was somewhat toxic as also noted
in the Yersinia infection model (cf. Fig. 2) while the most potent
compounds INP1767 and INP1855 result in cell viability >90%
(Table 2). The effect of INP1855 on growth of C. trachomatis was
studied by fluorescence microscopy after immunostaining
(Fig. 3). In absence of inhibitor large Chlamydia inclusions are
Figure 1. Dose-response curves for INP1750, 1767, and 1855 in the T3S reporter-
gene assay.
visible (Fig. 3, left panel). At 1.56
lM of INP1855 the size of the
inclusions are slightly reduced and at the MIC, 3.13
sions are visible and HeLa cell morphology is similar to the DMSO
treated control (Fig. 3, middle and right panels).
l
M, no inclu-
Table 2
EC50 values for the reporter-gene signal in Y. pseudotubercolosis, MIC for C. trachomatis
infection of HeLa cells, and HeLa cell viability for INP1750, INP1767, and INP1855
Cell viabilityb
a
Substance
EC50
C. trachomatis MIC
INP1750
INP1767
INP1855
12.4
14.6
6.24
25
12.5
3.13
66.3 5.4
92.1 2.4
90.2 4.0
a
a
Calculated from Figure 2 using GraphPad prism.
Percent cell viability ( SD) at 2Â MIC.
b
with EC50 values between 6 and 12
INP1855 did not affect bacterial growth at 100
l
M (Fig. 1, Table 2). INP1750 and
M and INP1767
M for periods up to 6 h (data not shown).6
l
showed no effect at 50
l
The effect of the compounds in the bacterial reporter-gene assay
was unaffected in presence of detergent indicating that compound
aggregation is not an underlying reason for the observed activity
(data not shown).24 This compound class clearly constitutes puta-
tive T3S inhibitors that either target the secretion machinery di-
rectly or act on regulatory pathways.25
b
c
From the data in Table 1 some conclusions regarding structure–
activity relationships can be drawn. It is clear that the fused pyri-
dine ring is critical since INP1759 and INP1758, that lack the ring
completely, and the naphthalene based INP1853 are void of activ-
ity. The aromatic hydroxyl group appears to be preferred as the
corresponding methyl ethers INP1869 and INP1870 are inactive.
The nitro group in INP1750 can be exchanged for a bromo or chloro
substituent but when comparing INP1750 with INP1813 and
INP1855 with INP1788 it is obvious that the nitro group is supe-
rior. INP1860 that lacks a substituent in this position still shows
moderate activity at higher concentrations but it was found that
this effect most likely results from general growth inhibition (data
not shown). Modification of the N-phenyl piperazine moiety in
INP1750 can be executed resulting in retained or improved activity
as shown by INP1767 and INP1855. INP1767 was tested in racemic
form and it remains to be established if the individual stereoiso-
mers display different biological profiles.
Figure 2. Effect of INP1750, INP1767, and INP1855 on T3S mediated virulence in a
macrophage infection model.6,26 Macrophage health was measured according to the
CalceinAM method. 100% on the y-axis corresponds to uninfected cells treated with
DMSO alone. (a) Wild-type Y. pseudotuberculosis (pIB102), (b) T3S deficient mutant
(pIB604), (c) uninfected control.
The three most promising compounds INP1750, INP1767, and
INP1855 were further evaluated in a macrophage infection model
as described previously.6,26 The macrophage cell line J774 was in-

P.-A. Enquist et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3550–3553
3553
Figure 3. Effect of INP1855 on growth of C. trachomatis in HeLa cells.6,26 The HeLa cells are stained red and the C. trachomatis inclusions are stained green (pathfinder
chlamydia, bio-rad).
luminescence signal was recorded with
reader.
21. Tramontini, M. Synthesis 1973, 12, 703.
a conventional filter-based plate
In conclusion we have employed whole cell screening to
identify putative inhibitors of T3S in Gram-negative bacteria. The
identified inhibitors are based on a different chemical scaffold
than previously reported small molecule T3S inhibitors.29–35
Subsequent investigation of SARs resulted in inhibitors that show
low micromolar activity against both the extracellular pathogen
Y. pseudotuberculosis and the intracellular pathogen C. trachomatis.
22. INP1758, 1759, 1764, 1765, 1766, 1768, 1769, 1772, and 1788 were purchased
from ChemBridge. All other compounds were synthesized in house or by
Syngene International Ltd. Typical experimental procedure: All starting
materials and reagents were commercially available and used as received.
Formaldehyde (75 mmol) and the cyclic secondary amine (20 mmol) were
reacted under cooling on ice and the resulting hard white precipitation was
added portionwise to the 8-hydroquinoline derivative (20 mmol) dissolved in
60 mL pyridine under stirring at 50 °C. The added precipitate reacted readily
and after a couple of minutes a yellowish precipitation was formed. Aftert 30–
40 min the mixture was filtered through a Büchner funnel. The precipitate was
washed with 150 mL ethanol, dissolved in 1 M HCl under heating and
crystallized by addition of 1 M NaOH under gentle stirring until pH 5–6. The
crystals were washed with water and the isolated product was >95% pure
according to LC–MS and 1H NMR spectroscopy.
Acknowledgments
We thank the Swedish Research Council, the Swedish Govern-
mental Agency for Innovation Systems (VINNOVA), the Knut &
Alice Wallenberg foundation, and the Carl Trygger foundation for
support. AstraZeneca Sweden, the Medical faculty at Umeå Univer-
sity and the Västerbottens county council.
23. 5-Nitro-7-((4-phenylpiperazin-1-yl)methyl)quinolin-8-ol (INP1750): 1H NMR
(400 MHz, DMSO-d6): d 9.19 (dd, J = 1.4, 8.7 Hz, 1H), 8.72 (dd, J = 1.2, 4.1 Hz,
1H), 8.6 (s, 1H), 7.64 (dd, J = 4.1, 8.7 Hz, 1H), 7.24–7.20 (m, 2H), 6.95–6.93 (m,
2H), 6.82–6.78 (m, 1H), 4.1 (s, 2H), 3.72–3.35 (m, 4H), 3.12–3.10 (m, 4H). 13C
NMR (100 MHz, DMSO-d6): d 150.2, 147.1, 139.8, 132.5, 132.2, 129.0, 127.4,
124.6, 124.3, 119.3, 116.0, 115.8, 115.7, 55.7, 51.7, 46.7. LC-MS [MÀH+]À Calcd
for [C20H20N4O3]: m/z 363.15. Found 363.17. 5-nitro-7-((3-phenylpyrrolidin-1-
yl)methyl)quinolin-8-ol (INP1767): 1H NMR (400 MHz, DMSO-d6): d 9.31–9.29
(m, 1H), 8.65 (s, 1H), 8.58 (br s, 1H), 7.54 (br s, 1H), 7.33 (s, 4H), 7.24 (br s, 1H),
4.38–4.35 (m, 2H), 3.83–3.79 (m, 1H), 3.66–3.52 (m, 3H), 3.36–3.31 (m, 1H),
2.39 (m, 1H), 2.13–2.10 (m, 1H). 13C NMR (100 MHz, DMSO-d6): d 146.3, 140.8,
134.6, 132.6, 129.2, 129.1, 127.8, 127.6, 127.5, 127.1, 125.1, 122.7, 115.5. LC-
MS [MÀH+]À Calcd for [C20H19N3O3]: m/z 348.14. Found 348.19. 7-((4-(4-
fluorophenyl)piperazin-1-yl)methyl)-5-nitroquinolin-8-ol (INP1855): 1H NMR
(400 MHz, DMSO-d6): d 11.53 (br s, 1H), 9.31 (dd, 1.4, 8.9 Hz, 1H), 9.04 (dd,
1.4, 4.3 Hz, 1H), 9.01 (s, 1H), 8.00 (dd, 4.4, 8.9 Hz, 1H), 7.11–7.06 (m, 2H), 7.03–
7.00 (m, 2H), 4.58 (s, 2H), 3.70 (br s, 2H), 3.52 (br s, 2H), 3.30 (br s, 2H), 3.21 (br
s, 2H). 13C NMR (100 MHz, DMSO-d6): d 162.2, 156.8 (d, 237.3 Hz), 147.7, 146.2,
136.3, 134.7, 133.5, 132.3, 126.1, 123.6, 118.0 (d, 7.8 Hz), 115.6 (d, 22.4), 111.3,
52.5, 50.3, 46.2. LC-MS [MÀH+]À Calcd for [C20H19FN4O3]: m/z 381.14. Found
381.24.
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20. The screening was performed at Umeå Small Molecule Screening Facility that
currently is incorporated as the screening platform in Laboratories for
Chemical Biology Umeå. The screening was performed essentially as
described in Ref. 1 with the differences that the compound DMSO solutions
were transferred using
a 96-channel pipetting instrument and the