First-generation structure-activity relationship studies of 2,3,4,9-tetrahydro-1H-carbazol-1-amines as CpxA phosphatase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2019.05.003

Genetic activation of the bacterial two-component signal transduction system, CpxRA, abolishes the virulence of a number of pathogens in human and murine infection models. Recently, 2,3,4,9-tetrahydro-1H-carbazol-1-amines were shown to activate the CpxRA

Full text of DOI:10.1016/j.bmcl.2019.05.003

Accepted Manuscript
First-generation structure-activity relationship studies of 2,3,4,9-tetrahy-
dro-1H-carbazol-1-amines as CpxA phosphatase inhibitors
Yangxiong Li, Jessi J. Gardner, Katherine R. Fortney, Inga V. Leus, Vincent
Bonifay, Helen I. Zgurskaya, Alexandre A. Pletnev, Sheng Zhang, Zhong-Yin
Zhang, Gordon W. Gribble, Stanley M. Spinola, Adam S. Duerfeldt
PII:
S0960-894X(19)30289-6
https://doi.org/10.1016/j.bmcl.2019.05.003
BMCL 26423
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 March 2019
2 May 2019
3 May 2019
Please cite this article as: Li, Y., Gardner, J.J., Fortney, K.R., Leus, I.V., Bonifay, V., Zgurskaya, H.I., Pletnev,
A.A., Zhang, S., Zhang, Z-Y., Gribble, G.W., Spinola, S.M., Duerfeldt, A.S., First-generation structure-activity
relationship studies of 2,3,4,9-tetrahydro-1H-carbazol-1-amines as CpxA phosphatase inhibitors, Bioorganic &
Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.05.003
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First-generation structure-activity relationship studies of 2,3,4,9-tetrahydro-1H-carbazol-1-amines
as CpxA phosphatase inhibitors
Yangxiong Li,a,b,† Jessi J. Gardner,a,b, † Katherine R. Fortney,c Inga V. Leus,b Vincent Bonifay,b Helen I.
Zgurskaya,b Alexandre A. Pletnev,d Sheng Zhang,e Zhong-Yin Zhang,e,f,g Gordon W. Gribble,d Stanley M.
Spinola,c,h,i Adam S. Duerfeldta,b*
aInstitute for Natural Products Applications and Research Technologies and bDepartment of Chemistry &
Biochemistry, Stephenson Life Sciences Research Center, Norman, Oklahoma 73019-5251
c
e
h
i
Departments of Microbiology and Immunology, Biochemistry, Medicine, Pathology and Laboratory
Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202
dDepartment of Chemistry, Dartmouth College, Hanover New Hampshire, 03755
fDepartment of Medicinal Chemistry and Molecular Pharmacology and the gInstitute for Drug Discovery,
Purdue University, West Lafayette, IN 47907
ABSTRACT
Genetic activation of the bacterial two component signal transduction system, CpxRA, abolishes the
virulence of a number of pathogens in human and murine infection models. Recently, 2,3,4,9-tetrahydro-
1H-carbazol-1-amines were shown to activate the CpxRA system by inhibiting the phosphatase activity of
CpxA. Herein we report the initial structure-activity relationships of this scaffold by focusing on three
approaches 1) A-ring substitution, 2) B-ring deconstruction to provide N-arylated amino acid derivatives
and 3) C-ring elimination to give 2-ethylamino substituted indoles. These studies demonstrate that the A-
ring is amenable to functionalization and provides a promising avenue for continued optimization of this
chemotype. Further investigations revealed that the C-ring is not necessary for activity, although it likely
provides conformational constraint that is beneficial to potency, and that the (R) stereochemistry is
required at the primary amine. Simplification of the scaffold through deconstruction of the B-ring led to
inactive compounds, highlighting the importance of the indole core. A new lead compound 26 was
identified, which manifests a ~30-fold improvement in CpxA phosphatase inhibition over the initial hit.
Comparison of amino and des-amino derivatives in bacterial strains differing in membrane permeability
and efflux capabilities demonstrate that the amine is required not only for target engagement but also for
permeation and accumulation in Escherichia coli.
Keywords: two-component system, sensory kinase, antibacterial, drug discovery, medicinal chemistry,
CpxRA, permeability, efflux
*Corresponding author
Email address: adam.duerfeldt@ou.edu (A.S. Duerfeldt)
†authors contributed equally
The urgency to develop new modalities to treat drug-resistant infections is unquestionable,
especially for those caused by multi- and extensively-drug resistant Gram-negative pathogens. Reports of
infectious strains resistant to all known clinically approved antibiotics have surfaced and raise the

possibility that common infections may soon become untreatable if new therapeutic advancements are not
developed in a timely fashion.1
All currently available antibiotics inhibit a limited number of enzymes and processes that have
essential functions for bacterial growth.2 The dogma in the field is that only compounds that completely
inhibit cell growth will prove to be clinically useful. Despite intensive efforts, few new targets or drug
classes have been identified for Gram-negative bacteria in the past 50 years.3 In fact, the ability to address
resistance has resorted in large part to structural modification of known classes.4 This approach typically
provides only short-lived solutions, as new derivatives are susceptible to the rapid evolution of cross-
resistance.5 The reality is that bacterial resistance now outpaces our ability to derivatize known scaffolds
and advance the new entities to the clinic. Thus, new targets, antibacterial chemotypes, and approaches
are desperately required. In contrast to traditional approaches, one strategy that is gaining significant
traction is to render organisms vulnerable to host immune clearance by targeting virulence determinants
or processes that control the expression of virulence factors.6
In this context, bacterial two-component signal transduction systems (2CSTS) represent attractive
targets for antibacterial drug discovery. In contrast to 2CSTS, that act usually at histidine and aspartate
residues, mammalian signal transduction systems utilize kinases and phosphatases that act at tyrosine,
serine, or threonine residues.7 Because 2CSTS are ubiquitous in bacteria, are not found in mammals, and
perform phosphorelay on different amino acids, they are excellent antimicrobial targets. These systems
consist of a sensory kinase (SK) and a response regulator (RR). In response to a stimulus (1, Figure 1),
the SK autophosphorylates a conserved histidine (2, Figure 1), and a cognate RR catalyzes the transfer of
this phosphate group to itself on an aspartic acid residue (3, Figure 1).8 Phosphorylation of the RR leads
to a biological response, usually resulting in changes to gene transcription. Some SKs also possess
phospho-RR phosphatase activity (4, Figure 1) and are thus critical to signaling duration.7 Loss of SK
phosphatase activity leads to persistent RR phosphorylation and thus constitutive activation of the
pathway.

Figure 1. CpxRA signaling overview.9-11 In response to membrane stress (1), CpxA (SK) undergoes autophosphorylation at a
conserved His residue in an ATP-dependent manner (2). CpxR (RR) then catalyzes the transfer of the phosphate group to a
cognate Asp residue (3), resulting in CpxR-P and activation. CpxA exhibits phosphatase activity and can dephosphorylate CpxR-
P to deactivate the system (4).
CpxRA, (SK = CpxA; RR = CpxR) is a 2CSTS found in many drug-resistant Gram-negative
bacteria, including all members of the Enterobacteriaceae, Neisseria gonorrhoeae, and Haemophilus
ducreyi. A major function of CpxRA is to alleviate membrane stress by reducing protein traffic through
the cell membrane.8 Activation of CpxRA reduces the expression of virulence factors, which must
traverse the cytoplasmic membrane to reach the cell surface. Uncontrolled, genetic activation of CpxRA
abolishes the virulence of Salmonella enterica serovar Typhimurium in mice.12 Additionally, genetic
activation of CpxRA by deletion of cpxA and loss of phosphatase activity abolishes the ability of H.
ducreyi to cause disease in human volunteers,13 of N. gonorrhoeae to infect mice,14 and impairs the ability
of uropathogenic Escherichia coli (UPEC) to cause pyelonephritis in mice.15 Thus, activation of CpxRA
impairs the ability of multiple extracellular and intracellular pathogens to infect their experimental or
natural hosts.
Most of what is known about 2CSTS has been produced through genetic manipulation of the
pathway constituents or by the development of a few small molecule inhibitors of these systems.16-17 As
such, the development of chemical probes to selectively modulate these systems is a valuable pursuit, as
these tools could be used to advance the understanding of these pathways and provide a better picture of
therapeutic utility. Recently, compound 1 (Figure 2) was shown to inhibit the phosphatase activity of

CpxA, resulting in accumulation of CpxR-P and thus activation of the CpxRA system (Figure 1) as
measured by a phospho-CpxR sensitive lacZ reporter in E. coli cells.18 Herein, we report our initial
progress in advancing the structure-activity relationships (SAR) for this 2,3,4,9-tetrahydro-1H-carbazol-1-
amine scaffold.
Figure 2. Initial SAR reported on the 2,3,4,9-tetrahydro-1H-carbazol-1-amine chemotype.18
Results reported in the initial discovery paper indicate that the 6-nitro functionality is not required
for CpxA phosphatase inhibition (2, Figure 2), but that removal of the primary amine abolishes activity
(3, Figure 2). To advance the SAR on this chemotype, we focused on three major approaches to reveal
structural motifs important for CpxA inhibition: 1) A-ring substitution, 2) B-ring deconstruction to
provide N-arylated amino acid derivatives and 3) C-ring removal to give 2-alkylamino substituted indoles
(Figure 3). Compounds were initially evaluated for their ability to activate a CpxR-responsive lacZ
reporter in a modified E. coli strain.18
O2N
C
R
A
B
NH2
NH2
NH2
N
H
N
N
H
H
A-ring
1
C-ring
substiitution
removal
O
R
N
H
NH2
B-ring
deconstruction
Figure 3. SAR strategy and rationale for the targeted analogs.
A-Ring Substitution. Due to previous observations that substitution is allowed on the 5-position
of the indole and may provide a means to alter the potency of this class, we evaluated a number of

substituents that ranged broadly in size, polarity, and hydrogen bonding capabilities. Analogs were
prepared in straight forward fashion via a two-step process employing Fischer-indole methodology19
followed by reductive amination (Scheme 1). As shown in Figure 4, the presence of a chlorine or
fluorine at the 5-position (5-6) provided the best activity, favoring the smaller, fluorine (6). While
incorporation of bromine at this position was also evaluated, evidence of bacterial cytotoxicity and
general activation of the stress response in the reporter strain was noted and thus the level of CpxRA
activation could not be determined. Installation of a trifluoromethyl group at the 5-position (7) also
provided equipotency to compound 1. Incorporation of more electron rich substituents like a methoxy (8)
or methyl (9) group was detrimental to activity. Inclusion of various other resonance mediated electron
withdrawing groups such as a methyl ester (10), carboxylic acid (11), sulfonamide (12), or nitrile (13)
failed to induce reporter activity beyond baseline, indicating no CpxA phosphatase inhibition for these
analogs.
O
a
b
O
R
R
NH2
R
N
O
NH2
N
N
H
H
H
i
ii
iii
iv
Scheme 1. General synthetic approach to 1–13. Reagents and conditions: (a) 1 M HCl, H2O, 120 °C, 32-66%; (b) NH4OAc,
NaCNBH3, MeOH, 60 °C, 73-94%.
In hopes of revealing activity trends based on basic physicochemical Hansch parameters (e.g.,
lipophilicity (π), size (Es), and electronics (σp)), –log (EC50) values were plotted versus individual
descriptors. Unfortunately, no obvious linear free energy relationship correlations are observed.

a
Figure 4. β-galactosidase activity of A-ring substituted analogs. Evidence of stress response activation due to cytotoxicity.
Results are provided as the mean ± SEM of at least three separate experiments performed in triplicate. ND = not determined
To further the interrogation of A-ring substitution, we performed a “substituent walk” to evaluate
the optimal positioning for the fluoro and trifluoromethyl substituents. The observation that the 6-fluoro
analog 16 exhibited near equipotency to 6 led us to synthesize and evaluate the 5,6-difluoro analog 20
(Figure 5). Gratifyingly, this substituent combination proved to be additive and improved activity ~3-fold
from compound 6 and ~5-fold from compound 16 (Figure 5). Overall, in comparison to the initial hit 1
and the unsubstituted derivative 2, compound 20 exhibits ~9-fold and ~11-fold improvements in cellular
potency, respectively.

a
b
Figure 5. Results of “substituent-walk” around the A-ring. Low levels of agonism did not allow for EC50 calculation. General
activation of stress response due to cytotoxicity. Results are provided as the mean ± SEM of at least three separate experiments
performed in triplicate.
C-Ring Deconstruction. In parallel to the A-ring studies, two derivatives lacking the intact C-
ring were assessed for CpxRA agonism and thus provide insight into the importance of this feature. As
shown in Figure 6, simplification of the 2,3,4,9-tetrahydro-1H-carbazol-1-amine scaffold to provide
compound 21 resulted in an apparent decrease in bacterial cytotoxicity, as no evidence of cell death was
observed at the concentrations evaluated, thus allowing for calculation of an EC50 value. . Comparison of
6 and 22, reveals a 10-fold loss in activity when the C-ring is eliminated. These initial results suggest that
the C-ring is not necessary for target engagement, but likely provides beneficial conformational bias that
improves ligand-target engagement. It is also worth noting that 21 showed no signs of general activation
of the stress response, a complication observed for the related compound, 4.

Figure 6. Effects of C-ring deconstruction. Results are provided as the mean ± SEM of at least three separate experiments
performed in triplicate.
In addition to C-ring deconstruction, we were interested in probing the requirements associated
with the primary amine. As mentioned previously, initial studies revealed the amine to be essential for
activity (Figure 2). It was not known, however, if stereochemical preference exists at this position and/or
if the amino group was simply providing hydrogen bond interactions that could be replenished with
different functionalities. To probe these questions, we synthesized 23-25 (Figure 7). Access to
enantiopure amines was achieved through the use of Ellman’s sulfinamides as chiral auxillaries to induce
stereoselective reductive amination (Scheme 2). As shown in Figure 7, only one of the two enantiomers
is active, with the (R)-enantiomer 23 being eutomer and the (S)-enantiomer 24 the distomer.
b
c
a
O
R
O
*
R
R
R
S*
*
S*
N
HN
N
N
O
NH2
N
N
H
H
H
H
iii
v
vi
vii
Scheme 2. Synthesis of compounds 23 and 24. Reagents and conditions: (a) t-butanesulfinamide, Ti(OEt)4, toluene, reflux, 84%;
(b) NaBH4, THF, -78 °C; (c) HCl▪dioxane, MeOH, 23 °C 99%.
Additionally, replacement of the primary amine with an alcohol produces an analog that is
inactive up to 100 µM in the β-galactosidase assay (25, Figure 7). As demonstrated by 26, incorporation
of the 5,6-difluoroindole with the (R) stereocenter produces the same enhancement in activity as noted
with the racemic series and provides the most active compound identified in this series to date.

Figure 7. Assessment of amine substitution and stereochemical requirements. Results are provided as the mean ± SEM of at least
three separate experiments performed in triplicate.
Considering the seemingly dispensable C-ring, the requirement for a primary amine, and
necessity of (R) stereochemistry, we were intrigued by the possibility of simplifying this scaffold even
further through the deconstruction of the B-ring to arrive at more synthetically tractable N-arylated amino
acids. As shown in Figure 3 this chemotype keeps the requisite A-ring and maintains the sp2
hybridization of the carbon alpha to the “indole” nitrogen. The resulting amide is expected to adopt the
more energetically favored trans geometry,20 thus providing a reasonable isostere for the parent indole,
while alleviating the rigidity of the parent heterocycle. Unfortunately, all analogs synthesized in this
series (Figure 8) were inactive at concentrations up to 80 µM.
F
F
F
O
O
O
N
H
N
N
H
H
NH2
NH2
O
NH2
O
27
28
29
F
F
F
F
F
O
N
N
N
H
H
H
NH2
NH2
NH2
30
31
32
Figure 8. seco-amide analogs synthesized and assessed for CpxRA modulation.
As mentioned previously, 1 is believed to indirectly activate the CpxRA 2CSTS through the
inhibition of CpxA phosphatase activity, thus rendering CpxR phosphorylated for an indefinite period of
time.18 To confirm whether the results of the lacZ reporter assay observed for the more potent derivatives

were consistent with mechanisms reported for 1, we evaluated the ability of 23 to inhibit CpxA
phosphatase activity through Phos-Tag methodology, gel visualization, and densitometry quantification of
CpxR phosphorylation levels, exactly as described.18 As is seen Figure 9 and as reported previously,18
both the ΔcpxA mutant and the compound-treated wild type expressed more CpxR than the untreated wild
type, likely because CpxR-P positively autoregulates its transcription. Similar to compound 1, treatment
with compound 23 (10 uM) caused a statistically significant increase in the ratio of CpxR-P to CpxR
relative to the untreated wild type and the ΔcpxA mutant. In contrast, activation in the ΔcpxA mutant
increased the accumulation of CpxR-P but did not cause a statistically significant increase in the ratio of
CpxR-P to CpxR compared to the wild type. Taken together, the data suggest that pharmacological
activation of CpxRA by inhibition of CpxA phosphatase activity yields different results than activation
via deletion of cpxA, perhaps because CpxA kinase activity is retained in the pharmacological approach.
Figure 9. Compound 23 induces phospho-CpxR accumulation. A) Composite representative Western blot of His6CpxR incubated
with 0 or 20 mM AcP, and the ΔcpxA mutant and WT grown in media with 0.4% glucose and 0 or 10 μM compound. Note that

migration of His6CpxR is slower than native CpxR. B) The ratio CpxR-P to CpxR was determined using densitometry; average
and standard deviation are from 5 independent experiments. P-values were calculated with one-way ANOVA.
The dependence of compound activity on the stereochemistry of the primary amine is indicative
of interaction with a 3-dimensional binding pocket within CpxA. Additionally, our SAR indicates that the
amine is essential for activity. In light of the recent work by the Hergenrother lab21 that highlights the
importance of primary amines in porin mediated transport, we were curious if the primary amine was also
involved in permeation and/or accumulation of the 2,3,4,9-tetrahydro-1H-carbazol-1-amine chemotype in
Gram-negative bacteria. To evaluate this, we developed a mass spectrometry method to determine the
accumulation of compounds 6 (a derivative from our studies) and 33 (a commercially available des-amino
comparator) in isogenic strains of E. coli with different efflux efficiencies and controlled permeability of
the outer membrane (Figure 10). WT-Pore cells contain the wild-type repertoire of efflux pumps and the
arabinose-controlled large pore that when expressed, permeabilizes the outer membrane to antibiotics as
WT
2000
1800
1600
1400
1200
1000
800
2000
1800
1600
1400
1200
1000
800
WT-pore
ΔTolC
B
A
ΔTolC-pore
F
600
600
400
400
NH2
200
200
N
H
0
0
0
10
20
30
40
50
60
70
80
90
0
10
20
30
40
50
60
70
80
90
6
[Out] (µM)
[Out] (µM)
400
350
300
250
200
150
100
50
400
350
300
250
200
150
100
50
WT
C
D
WT-pore
ΔTolC
ΔTolC-pore
F
N
H
0
0
0
10
20
30
40
50
60
70
80
90
0
10
20
30
40
50
60
70
80
90
[Out] (µM)
[Out] (µM)
33
Figure 10. Intracellular uptake of compounds 6 and 33 into isogenic E. coli strains that exhibit different efflux efficiencies
and controlled permeability of the outer membrane. WT = E.coli BW25113; WT-Pore = BW25113 attTn7:: attTn7::mini-
Tn7T-Kmrr -araC-ParaBAD-fhuAC4L ; ΔTolC = BW25113 tolC-ygiBC; ΔTolC-Pore = ΔTolC attTn7::mini-Tn7T-Kmr -
araC-ParaBAD-fhuAC4L.22

large as vancomycin.22 The ΔTolC cells are efflux-deficient derivatives lacking the outer membrane
channel TolC required for the activity of various efflux pumps of E. coli. Cells with intact and
hyperporinated (induced) outer membranes were incubated with increasing concentrations of compounds
6 and 33 and the intracellular accumulation was analyzed after 1 and 40 minutes of incubation with
compounds. As seen in Figure 10, compound 33, which lacks the primary amine, is susceptible to efflux
and suffers from poor permeability across the outer membrane. Compound 6 on the other hand contains
the primary amine, accumulates at much higher intracellular levels overall, is still sensitive to efflux,
albeit to a lesser extent, and accumulates less in wild-type than in efflux deficient E. coli. Additionally, 6
accumulates equally well in the cells with intact and hyperporinated outer membrane. These results
clearly demonstrate that the primary amine is not only involved in target engagement but is also
imperative to the permeation and accumulation in a Gram-negative organism.
Given that conventional approaches to develop new antibiotics have yielded few new drugs, the
potential reward of exploring novel targets and mechanisms is very high. Bacterial 2CSTS are major
players in organismal pathogenicity, fitness, communication, biofilm formation, and antibacterial
resistance making them attractive targets. Unfortunately, most of what we know about these systems has
been revealed through genetic manipulation or small molecule inhibitors of such systems, which is not
always indicative of therapeutic potential. As such, there is an increasing interest in developing small
molecule probes that can be used to activate these systems to uncover new biology that may translate to
new antibacterial approaches. Along these lines, we aimed to improve the activity of 1, a newly identified
CpxA phosphatase inhibitor that indirectly activates the CpxRA signaling pathway through increasing the
lifetime of CpxR-P. Leveraging three different structural modification approaches, we identified
compound 26 (EC50 = 1.1 µM), which exhibits ~30-fold improvement in CpxA phosphatase inhibition
over the initial hit 1 as determined in a cellular lacZ reporter assay. The stereochemical and
conformational requirements found for the primary amine of this chemotype illustrates its importance in
target engagement and involvement in membrane permeation and protection against efflux in E. coli.
Studies are ongoing to determine the CpxRA selectivity (or lack thereof) of this chemotype and to assess

its efficacy in animal models of infection. Nonetheless, this work provides useful SAR and a foundation
for which to advance the utility of 2,3,4,9-tetrahydro-1H-carbazol-1-amines as chemical probes to
uncover new biology in bacterial two-component signal transduction systems.
ACKNOWLEDGEMENTS
Research reported in this publication was supported by the National Institute of Allergy and Infectious
Disease of the National Institutes of Health under award number 1R01AI136795 (A.S.D. and H.I.Z), by a
Project Development Team within the Indiana CTSI NIH/NCRR Grant Number UL1TR001108 (SMS),
and by the Chemical Genomics Core facility in the Indiana CTSI. The content is solely the responsibility
of the authors and does not necessarily represent the official views of the National Institutes of Health.
SUPPLEMENTARY DATA
Supplemental data [synthetic procedures, biological methods, and characterization information for new
compounds 23, 24, and 26] to this article can be found online at xxxxxx.
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Table of Contents Graphic
First-generation structure-activity relationship studies of 2,3,4,9-tetrahydro-1H-carbazol-1-amines
as CpxA phosphatase inhibitors
Yangxiong Li,a,b,† Jessi J. Gardner,a,b, † Katherine R. Fortney,c Inga V. Leus,b Vincent Bonifay,b Helen I.
Zgurskaya,b Alexandre A. Pletnev,d Sheng Zhang,e Zhong-Yin Zhang,e,f,g Gordon W. Gribble,d Stanley M.
Spinola,c,h,i Adam S. Duerfeldta,b*