Discovery of niclosamide and its O-alkylamino-tethered derivatives as potent antibacterial agents against carbapenemase-producing and/or colistin resistant Enterobacteriaceae isolates

Article abstract of DOI:10.1016/j.bmcl.2019.03.032

Carbapenemase-producing Enterobacteriaceae (CPE) represents the most worrisome evolution of the antibiotic resistance crisis, which is almost resistant to most of available antibiotics. This situation is getting even worse particularly due to the recent emergence of colistin resistance. Herein, niclosamide, an FDA-approved traditional drug, and its novel O-alkylamino-tethered derivatives were discovered as new and potent antibacterial agents against carbapenemase-producing and/or colistin resistant Enterobacteriaceae isolates. Among these molecules, compound 10 (HJC0431) with 4-aminobutyl moiety showed the broad antibacterial activities, effective against 6 strains. In vitro checkerboard and time-kill course studies demonstrated the synergistic effects of the screened compounds with colistin against the corresponding strains with various degrees.

Full text of DOI:10.1016/j.bmcl.2019.03.032

Accepted Manuscript
Discovery of niclosamide and its O-alkylamino-tethered derivatives as potent
antibacterial agents against carbapenemase-producing and/or colistin resistant
Enterobacteriaceae isolates
Jimin Xu, María Eugenia Pachón-Ibá ñez, Tania Cebrero-Cangueiro, Haiying
Chen, Javier Sánchez-Céspedes, Jia Zhou
PII:
S0960-894X(19)30171-4
https://doi.org/10.1016/j.bmcl.2019.03.032
BMCL 26346
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
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18 March 2019
22 March 2019
Please cite this article as: Xu, J., Pachón-Ibá ñez, M.E., Cebrero-Cangueiro, T., Chen, H., Sánchez-Céspedes, J.,
Zhou, J., Discovery of niclosamide and its O-alkylamino-tethered derivatives as potent antibacterial agents against
carbapenemase-producing and/or colistin resistant Enterobacteriaceae isolates, Bioorganic & Medicinal Chemistry
Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.03.032
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Bioorganic & Medicinal Chemistry Letters
Discovery of niclosamide and its O-alkylamino-tethered derivatives as potent
antibacterial agents against carbapenemase-producing and/or colistin resistant
Enterobacteriaceae isolates
Jimin Xua,†, María Eugenia Pachón-Ibáñezb,†,Tania Cebrero-Cangueirob,†, Haiying Chena, Javier Sánchez-
Céspedesb,, and Jia Zhoua,*
a Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Blvd, Galveston, Galveston, Texas 77555, United States
b Unit of Infectious Diseases, Microbiology and Preventive Medicine, Institute of Biomedicine of Seville (IBiS), University Hospital Virgen del
Rocío/CSIC/University of Seville, E41013 Seville, Spain
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Carbapenemase-producing Enterobacteriaceae (CPE) represents the most worrisome evolution
of the antibiotic resistance crisis, which is almost resistant to most of available antibiotics. This
situation is getting even worse particularly due to the recent emergence of colistin resistance.
Herein, niclosamide, an FDA-approved traditional drug, and its novel O-alkylamino-tethered
derivatives were discovered as new and potent antibacterial agents against carbapenemase-
producing and/or colistin resistant Enterobacteriaceae isolates. Among these molecules,
compound 10 (HJC0431) with 4-aminobutyl moiety showed the broad antibacterial activities,
effective against 6 strains. In vitro checkerboard and time-kill course studies demonstrated the
synergistic effects of the screened compounds with colistin against the corresponding strains
with various degrees.
Keywords:
Niclosamide derivatives
Antibacterial agents
Carbapenemase-producing Enterobacteriaceae
Colistin resistance
2009 Elsevier Ltd. All rights reserved.
Synergistic effects
The development of antibiotics for the treatment of fatal
infections is considered one of the major breakthroughs of
modern medications. However, the acquisition of resistance
towards their antimicrobial activity has inevitably been
happening. The emergence and dissemination of antibiotic
resistant pathogens is becoming a serious threat to human health.
mechanistically different from each other, and this makes it
challenging to identify an agent that is active against all types of
CPE. Colistin and tigecycline have been reported as the
remaining therapeutic options against various CPE infections7-9.
Nevertheless, over the past few years, the colistin resistant CPE
cases have been sporadically reported from various parts of the
world, such as South Korea, Israel, Singapore and United
States10. Thus, there is an urgent need for the screening and
development of new antimicrobial agents to keep up with the
evolution of the drug resistance mechanisms in the
Enterobacteriaceae family.
The
“ESCAPE”
pathogens
(Enterococcus
faecium,
Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter
baumannii, Pseudomonas aeruginosa, and Enterobacter species)
are responsible for the majority of nosocomial infection and
capable of escaping the biocidal action of antimicrobial agents1-3.
In particular, carbapenemase-producing Enterobacteriaceae
(CPE, mostly contributed by K. pneumonia) represents the most
worrisome evolution of the antibiotic resistance crisis, which is
almost resistant to all available antibiotics, and the mortality rates
associated with CPE infections are intolerably high4, 5. Currently,
Niclosamide, an FDA-approved drug, has been traditionally
used in the clinic for the treatment of tapeworm infections for
several decades, and it is well tolerated with extremely high acute
oral LD50 values of > 1000 mg/kg11, 12. Its mechanism of
anticestodal action has been reported to involve uncoupling
the
clinically
most
important
carbapenemases
in
oxidative
phosphorylation
and
stimulating
adenosine
Enterobacteriaceae include the class A enzymes of the Klebsiella
pneumoniae carbapenemases (KPC) type, the zinc-dependent
class B metallo-β-lactamases (MβLs) of the VIM, IMP, and
NDM types and the plasmid-expressed class D carbapenemases
of the OXA-48 type6. These types of CPE are structurally and
triphosphatase activity in the mitochondrial12-15. Over the past
several years, accumulated studies showed that niclosamide is a
multifunctional drug that can regulate multiple signaling
pathways and biological processes including Wnt/β-catenin,
mTORC1, STAT3, NF-κB, Notch, NS2B-NS3 protease, pH,
———
 Corresponding authors. Jia Zhou, Tel.: +1-409-772-9748; fax: +1-409-772-9648; e-mail: jizhou@utmb.edu. Javier Sánchez Céspedes, Tel.:
+34 955 923 100; fax: +34 955 923 101; email: jsanchez-ibis@us.es.
†These authors contributed equally to the manuscript.

etc16-25. Due to its various biological properties, niclosamide has
the great potential being repurposed to treat other human
conditions, such as cancer, viral infections and tuberculosis, more
than just helminthic diseases17. To improve the poor aqueous
solubility and bioavailability of niclosamide, our group
discovered a series of O-alkylamino-tethered derivatives as
potent and orally bioavailable anticancer agents26. Herein we
reported the screening and studies of these niclosamide
derivatives with enhanced drug-like properties against different
clinical carbapenemase-producing and/or colistin (COL) resistant
Enterbacteriaceae isolates.
aminoethoxy) ethyl moiety also exhibited inhibitory activities
against two strains: E.coli NDM-1 and E. cloacae 297. However,
Compounds 7 with 2-aminoethyl moiety and 9 with 3-
aminopropyl were found to have no effects. The active
compounds 8 and 10-12 all bear a terminal amino side chain at
the phenol moiety significantly improving their aqueous
solubility, and the four carbon length side chain (10)
displayed good and broad antibacterial activities. Meanwhile,
compound 10 showed excellent aqueous solubility, with a
saturated concentration of 650 µg/mL, which was determined by
an HPLC analysis method30. Other amino derivatives 2-6 and 13-
14 with no terminal NH2 moiety displayed no inhibitory activities
against the sixteen selected strains at 50 µg/mL.
The synthesis of the selected compounds was summarized in
Scheme 1. Analogues 2-6 were easily prepared by Mitsunobu
reaction of niclosamide with corresponding amino alcohols.
Mitsounobu coupling of niclosamide with N-Boc-protected
amino alcohols was followed by Boc-deprotection to afford
compounds 7-14. The structures and purity of all synthesized
a
Scheme 1
1
compounds were confirmed by H and 13C NMR, HR-MS and
HPLC analysis26, 27. Sixteen clinical carbapenemase-producing
and/or colistin resistant (COLR) Enterbacteriaceae isolates were
selected for screening including carbapenemase-producing
Klebsiella pneumoniae (KPC) (4 strains), standard KPC
(CECT997), carbapenemase-producing NDM-1 COL-susceptible
(2 strains) and Enterobacter COL-resistant (9 strains). As shown
in Table 1, five compounds including niclosamide were
discovered with antibacterial activities at the concentration of 50
µg/mL28, 29. Niclosamide (1) showed inhibitory activities against
two COL resistant strains: E. cloacae 280 and E. cloacae 297.
Compound 8 with S-2-aminobutyl moiety inhibited the growth of
E.coli NDM-1. Excitingly, compound 10 with 4-aminobutyl
moiety showed the broad antibacterial activities, effective against
6 strains: KPC CECT-997, KPC-28, KPC NDM-1, E.coli NDM-
1, E. cloacae 280 and E. cloacae 297. Compound 11 with 5-
aminopentyl moiety was effective against KPC-28, KPC NDM-1
and E.coli NDM-1. Interestingly, compound 12 with 2-(2-
aReagents and conditions: (a) ROH, Ph3P, DIAD, THF, r.t, 2-6. (b) i. Boc-
ROH, Ph3P, DIAD, THF, r.t; ii. TFA, CH2Cl2, 0°C to r.t, 7-14.
Table 1. Inhibition screening of niclosamide and its O-aminoalkyl-tethered derivatives against selected clinical strains of
carbapenemase-producing Enterobacteriaceae isolates and / or colistin resistant a
KPC
E. aerogenes
E. cloacae
E.coli
NDM-1
KPC
NDM-1
Compd
CECT
07
-
21
-
28
-
29
-
3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
53
32
142
190
263
272
280
297
+
-
-997
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
2
-
-
-
-
-
-
-
-
+
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
4
-
-
-
-
-
-
-
-
5
-
-
-
-
-
-
-
-
6
-
-
-
-
-
-
-
-
7
-
-
-
-
-
-
-
-
8
-
-
-
-
+
-
-
-
-
9
-
-
-
-
-
-
-
10
11
12
13
14
-
-
+
+
-
-
+
+
+
-
+
+
-
+
-
+
-
-
-
-
-
-
-
-
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-

a
All compounds were screened at 50 µM; + Inhibition of bacterial growth; - No inhibition of bacterial growth; KPC: Klebsiella pneumoniae carbapenemases;
KPC-07 (VIM-1) (COL susceptible (S) MIC COL= 0.5 µg/mL); KPC-21 (VIM-1, DHAI) (COL resistant (R) MIC COL= 64 µg/mL); KPC-28 (OXA-48, CTX-
M-15) (COLS MIC COL= 0.5 µg/mL); KPC-29 (OXA-48, CTX-M-15) (COLR MIC COL= 32 µg/mL); KPC NDM-1 (MIC COL= 0.5 µg/mL); E.coli NDM-1
(MIC COL= 0.5 µg/mL); E. aerogenes 3 (MIC COL= 32 µg/mL); E. cloacae 32 (MIC COL > 256 µg/mL); E. aerogenes 53 (MIC COL= 32 µg/mL); E. cloacae
142 (MIC COL= 64 µg/mL); E. cloacae 190 (MIC COL= 32 µg/mL); E. cloacae 263 (MIC COL= 8 µg/mL); E. cloacae 272 (MIC COL= 16 µg/mL); E. cloacae
280 (MIC COL= 32 µg/mL); E. cloacae 297 (MIC COL= 3 µg/mL).
The minimal inhibitory concentration (MIC) values of the
selected in vitro active derivatives were further evaluated31, 32. As
listed in Table 2, the MIC values of compound 10 against KPC
CTCT-997, KPC-28 and E.coli NDM-1 were 15 µg/mL.
Compound 11 also showed good inhibitory activity against KPC-
28 strain with an MIC of 15 µg/mL, while all other MIC values
were higher than 15 µg/mL.
effects with COL against the selected strains. Some of the time-
kill curves also confirmed these synergistic effects. The
combination of niclosamide (1) and COL was bactericidal against
E. cloacae 280 since 2 hrs and synergistic since 8 hrs (Figure
1A). Compound 10 and COL showed bactericidal effect against
E. coli NDM-1 since 2 hrs, while their combination was
bactericidal since 2 hrs as well (Figure 1B). Compound 10 had a
synergistic effect with COL against KPC-28 since 8 hrs, and the
combination was bactericidal from 4 hrs to 8 hrs (Figure 1C).
The combination of 11 and COL also had a bactericidal and
synergistic activity against KPC-28 since 2 hrs (Figure 1D).
Table 2. Minimal inhibitory concentration (MIC; μg/mL) of the
in vitro active derivativesa
KPC
E. cloacae
E. coli
NDM-1
KPC
NDM-1
Compd
CECT-
997
28
-
280 297
Table 3. Checkerboard assay of the in vitro active derivativesa
Compd
Strains
Concentrations
FICI
1
-
-
-
>15 >15
E. cloacae 280
8
-
-
>15
15
-
-
-
1
1/8 COL+ 1/8 compound 1
0.25
E. cloacae 297
E. coli NDM-1
KPCb NDM-1
E. coli NDM-1
10
11
12
15
-
15
15
-
>15
>15
-
>15 >15
8
1/4 COL+ 1/4 compound 8
1/4 COL+ 1/4 compound 10
1/8 COL+ 1/8 compound 10
0.50
0.50
0.25
>15
>15
-
-
-
-
>15
10
11
a KPC: Klebsiella pneumoniae carbapenemases; - No inhibition.
1/4 COL+ 1/8 compound 11
1/8 COL+ 1/4 compound 11
Considering the drawbacks of colistin and other potential
active antibiotics in monotherapy, combination therapy has been
raised as an interesting strategy to overcome those limitations as
a single agent. In vitro synergy against CPE has been
documented with some agents such as colistin-rifampin and
colistin-tigecycline33, 34. We also investigated in vitro synergisms
of these active derivatives together with COL using checkerboard
assay and time-kill kinetic assays. As depicted in Table 3, these
active compounds displayed various degrees of synergistic
E. coli NDM-1
0.38
a The FICI was interpreted as follow: synergism = FICI ≤ 0.5; indifferent >
0.5 to ≤ 4; antagonism = FICI > 4. The experiments were performed in
triplicate.
b KPC: Klebsiella pneumoniae carbapenemases.
Figure 1. Time-kill curves. (A) Compound 1 (HJC0111) and COL alone and in combination against E. cloacae 280. (B) Compound 10 (HJC0431) and COL
alone and in combination against E. coli NDM-1. (C) Compound 10 (HJC0431) and COL alone and in combination against KPC-28 (OXA-48, CTX-M-15). (D)
Compound 11 (HJC0389) and COL alone and in combination against KPC-28 (OXA-48, CTX-M-15).

In summary, niclosamide and its novel O-alkylamino-tethered derivatives (8, 10, 11 and 12) were discovered with antibacterial
activities against carbapenemase-producing and/or colistin resistant Enterobacteriaceae isolates. Among these compounds, 10
(HJC0431) with 4-aminobutyl moiety showed the broad antibacterial activities, effective against 6 strains. In vitro checkerboard
and time-kill course studies demonstrated the synergistic effect of the screened compounds with COL against the corresponding
strains with various degrees. These promising data support the further optimization potential of O-alkylamino-tethered derivatives
as new and unique antibiotics against carbapenemase-producing and/or colistin resistant Enterobacteriaceae isolates.
Acknowledgements
This work was supported by UTMB Technology Commercialization Program, and Sanofi Innovation Awards (iAwards), John
Sealy Memorial Endowment Fund, and Institute for Translational Sciences (ITS) at UTMB, Plan Nacional de I + D + i 2013-2016
and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía,
Industria y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0009), cofinanced by
European Development Regional Fund “A way to achieve Europe”, Operative program Intelligent Growth 20142020, the Instituto
de Salud Carlos III, Proyectos de Investigación en Salud (PI15/00489) and Proyectos de Desarrollo Tecnológico en Salud
(DTS17/00130), the Spanish Adenovirus Network (AdenoNet, BIO2015/68990-REDT), and the program “Nicolás Monardes” (C-
0059-2018) Servicio Andaluz de Salud, Junta de Andalucía.
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1
27. Spectra data of the representative compound: (S)-2-(2-Aminobutoxy)-5-chloro-N-(2-chloro-4-nitrophenyl)benzamide (8). Pale yellow solid. H
NMR (600 MHz, DMSO-d6) δ 8.51 (d, J = 9.1 Hz, 1H), 8.43 (d, J = 2.4 Hz, 1H), 8.28 (dd, J = 9.1, 2.6 Hz, 1H), 7.92 (d, J = 2.8 Hz, 1H), 7.64
(dd, J = 8.9, 2.8 Hz, 1H), 7.38 (d, J = 9.0 Hz, 1H), 4.24 (dd, J = 10.2, 5.0 Hz, 1H), 4.06 (dd, J = 10.1, 7.5 Hz, 1H), 3.02 (s, 1H), 1.56 – 1.48 (m,
1H), 1.32 – 1.26 (m, 1H), 0.91 (t, J = 7.5 Hz, 3H). 13C NMR (150 MHz, DMSO-d6) δ 162.7, 155.6, 143.3, 141.2, 133.4, 130.4, 125.1, 124.8,

124.5, 123.4, 123.3 (2C), 116.4, 74.8, 51.0, 26.5, 10.1. HRMS (ESI) calcd for C17H18Cl2N3O4 398.0674 (M + H)+, found 398.0670. HPLC
analysis conditions: Waters μBondapak C18 (300 × 3.9 mm); flow rate 0.5 mL/min; UV detection at 270 and 254 nm; linear gradient from 10%
acetonitrile in water (0.1% TFA) to 100% acetonitrile (0.1% TFA) in 20 min followed by 30 min of the last-named solvent; tR = 17.07 min; purity
(by peak area) 96%.
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Pharmacologically Active Small Molecules against Staphylococcus aureus Biofilms. Antimicrob Agents Chemother. 2016; 60(10): 5663-5672.
29. The activity screening studies were performed as follows: All compounds were screened at 15 µg/mL in 96-well plates to identify which
produced inhibition of growth of the 16 clinical Enterobacteriaceae strains. Briefly, 50 µL of 1 × 106 Colony Forming Unit (CFU) were
inoculated in 50 µL Müeller Hinton Broth (MHB) medium in each well containing the different derivatives. Then, plates were incubated at 37 °C
with humidity for 18 hrs. For each tested compound and strain, if after the incubation visual growth was observed in the wells the compound
activity was considered negative. If no visual growth was observed in the wells the compound activity was considered positive. The screening
was performed in duplicate.
30. Vogel, H. G.; Hock, F. J.; Maas, J..Mayer, D. Determination of solubility by hyphenated HPLC methods. Drug discovery and evaluation: safety
and pharmacokinetic assays. New York: Springer; 2006: 400-402.
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Aminoglycosides, Tigecycline, Rifampicin, and Doripenem Against Clinical Strains of Carbapenemase-Producing and/or Colistin-Resistant
Enterobacteriaceae. Front Cell Infect Microbiol. 2018; 8: 363.
32. For the determination of the Minimal Inhibitory Concentration (MIC) a broth microdilution method was used (cation-adjusted Müeller Hinton
broth; Becton Dickinson, Cockeysville, Md.), in accordance with the CLSI guidelines (Clinical and Laboratory Standards Institute, 2012). The
compounds for which inhibition of the bacterial growth was observed in the screening studies were tested (6 clinical strains). The MIC value was
the lowest concentration of derivative compound that completely inhibited the bacterial growth. All assays were performed in triplicate to ensure
reproducibility.
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Discovery of niclosamide and its O-
alkylamino-tethered derivatives as potent
antibacterial agents against carbapenemase-
producing and/or colistin resistant
Enterobacteriaceae isolates
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Jimin Xu, María Eugenia Pachón-Ibáñez,Tania Cebrero-Cangueiro, Haiying Chen, Javier Sánchez-Céspedes,
and Jia Zhou