Discovery of O-alkylamino-tethered niclosamide derivatives as potent and orally bioavailable anticancer agents

Article abstract of DOI:10.1021/ml3003082

Niclosamide has been identified to potently inhibit the activation, nuclear translocation, and transactivation of STAT3. Nevertheless, the poor aqueous solubility and bioavailability of niclosamide have hindered its further clinical development for cancer therapy. To discover new molecules with enhanced druglike properties, a series of novel O-alkylamino-tethered derivatives of niclosamide have been designed, synthesized, and biologically evaluated. Among them, compound 11 (HJC0152) has been demonstrated to significantly suppress MDA-MB-231 xenograft tumor growth in vivo (ip and po), indicating its great potential as efficacious and orally bioavailable therapeutics for human cancer.

Full text of DOI:10.1021/ml3003082

Letter
pubs.acs.org/acsmedchemlett
Discovery of O‑Alkylamino-Tethered Niclosamide Derivatives as
Potent and Orally Bioavailable Anticancer Agents
Haijun Chen,†,§ Zhengduo Yang,‡,§ Chunyong Ding,† Lili Chu,‡ Yusong Zhang,‡ Kristin Terry,‡
Huiling Liu,† Qiang Shen,*,‡ and Jia Zhou*,†
†Chemical Biology Program, Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas
77555, United States
‡Department of Clinical Cancer Prevention, Division of Cancer Prevention and Population Sciences, The University of Texas M. D.
Anderson Cancer Center, Houston, Texas 77030, United States
S
* Supporting Information
ABSTRACT: Niclosamide has been identified to potently inhibit the activation, nuclear
translocation, and transactivation of STAT3. Nevertheless, the poor aqueous solubility and
bioavailability of niclosamide have hindered its further clinical development for cancer
therapy. To discover new molecules with enhanced druglike properties, a series of novel O-
alkylamino-tethered derivatives of niclosamide have been designed, synthesized, and
biologically evaluated. Among them, compound 11 (HJC0152) has been demonstrated to
significantly suppress MDA-MB-231 xenograft tumor growth in vivo (ip and po), indicating
its great potential as efficacious and orally bioavailable therapeutics for human cancer.
KEYWORDS: niclosamide derivatives, STAT3, water solubility, orally bioavailable
ignal transducers and activators of transcription (STATs)
Nevertheless, niclosamide does not have an ideal pharmacoki-
Sare a family of transcription factors that serve as signaling netic profile due to its poor water solubility and low oral
bioavailability.23,24 It was of our interest to utilize rational
transmitters for a large number of cytokines and growth factors
chemical approaches to generate and identify novel derivatives
of niclosamide with improved aqueous solubility and
bioavailability as potential clinical candidates for cancer therapy.
Herein, we report our chemical structural modifications of
niclosamide to discover novel molecules aiming at enhanced
efficacy and improved druglike properties.
in the regulation of critical biological processes including cell
growth, proliferation, differentiation, and survival.1−6 Accumu-
lating evidence has demonstrated that persistent activation of
STAT3 stimulates tumor angiogenesis, promotes tumor
immune evasion, and even confers resistance to apoptosis
induced by conventional therapies.7−13 Therefore, STAT3
represents an attractive therapeutic target for the treatment of
various types of human cancer.14−16
We first directed our optimization effort involving
modification of the hydroxyl group on the phenol ring of
niclosamide by introduction of an O-alkylamino side chain. The
amino group-containing scaffolds are important motifs of
structural tuning with the capability to form hydrogen bonding
like the phenol group for selective protein binding and
druglikeness enhancement.25 These new O-alkylamino tethered
analogues were expected to provide efficient target binding for
a better potency, and as free bases, these molecules could also
form salts for the final target compounds to have a better
aqueous solubility. As shown in Scheme 1, analogues 2−6 were
conveniently prepared by Mitsunobu reaction. Alkylation of the
bromide intermediate 2 with a variety of amines including
heterocyclic moieties introduced basic functionalities into the
molecules, providing niclosamide derivatives 7 and 8.
Mitsunobu coupling of niclosamide with N-Boc-protected
amino alcohols followed by the Boc deprotection afforded
analogues 9−15 with diversified O-alkyamino side chains. Both
Despite significant advances in recent discovery efforts
targeting STAT3, only several STAT3 inhibitors are advanced
into early phase clinical trials.16 A poor physicochemical
property is one of the most significant obstacles in promoting
a potent agent into clinical trials.16 For example, peptide and
peptidomimetics inhibitors targeting STAT3 suffer from poor
cellular permeability and stability.17−20 Aqueous solubility plays
an essential role in drug disposition and is one of the crucial
molecular properties for successful drug development.
Although an appropriate formulation could be used to enhance
solubility and absorption, the stability and manufacturing
difficulties should also be taken into consideration. In addition,
oral chemotherapy is obviously a preferred administrative route
in cancer treatment due to its convenience, patient compliance,
and cost effectiveness.21,22 Niclosamide, an FDA-approved
anticestodal drug, has recently been identified with significant
inhibition of the activation, nuclear translocation, and trans-
activation of STAT3.19 It inhibits the transcription of STAT3
target genes and induces cell growth inhibition, apoptosis, and
cell cycle arrest of cancer cells with constitutively active STAT3.
Received: October 2, 2012
Accepted: January 15, 2013
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
a
Scheme 1
Table 1. Effects of New Niclosamide Analogues on
Proliferation of Human Breast and Pancreatic Cancer Cell
Lines
IC50 (μM)
breast cancer
pancreatic cancer
a
compd
cLogP
MCF-7
1.06
>10
4.11
>10
9.87
>10
>10
12.4
0.25
MDA-MB-231
AsPC1
1.47
Panc-1
1
3
4.05
4.26
4.22
3.77
3.47
4.73
3.56
3.20
3.91
3.01
3.33
3.76
4.15
2.87
2.95
3.13
0.79
>10
2.69
>10
18.0
>10
>10
8.7
1.73
ND
>10
ND
>10
ND
ND
ND
0.54
1.08
3.11
>10
4.67
>10
ND
ND
b
ND
4
3.51
ND
72.2
ND
ND
ND
2.76
1.9
5
6
7
8
9
10
11
12
13
14
15
19
20
0.29
1.64
2.61
2.37
3.1
0.91
3.11
3.7
4.2
7.4
3.49
9.37
2.08
>10
ND
ND
5.3
>10
>10
>10
5.87
a
a
b
Reagents and conditions: (a) R1OH, Ph3P, DIAD, THF, rt, 50−93%.
cLogP: http://146.107.217.178/lab/alogps/start.html. ND, not
determined.
(b) R2H, KI, K2CO3, acetone, reflux, 55−91%. (c) (i) Boc-R1OH,
Ph3P, DIAD, THF, rt; (ii) TFA, CH2Cl2, 0 °C to rt, 34−81% (two
steps).
a
Scheme 2
linear alkyl amines and heterocyclic alkyl amines have been
explored for the purpose of comparison.
To examine whether the substitution of moiety groups
affected biological activities of newly synthesized analogues and
explore the structure−activity relationship (SAR), we first
evaluated the in vitro anticancer effects of the compounds 3−
15 on the proliferation of breast cancer cell lines MCF-7 (ER-
positive) and MDA-MB-231 (ER-negative and triple-negative),
as well as pancreatic cancer cell lines AsPC1 and Panc-1 using
MTS assays. The ability of these new analogues to inhibit the
growth of cancer cells is summarized in Table 1. The results
revealed that analogues 10−15 bearing the terminal amino
group-containing side chain at the phenol moiety showed
promising antiproliferative activities with low micromolar to
nanomolar IC50 values, while the alkyl-substituted derivatives
5−9 at the terminal amino group on the same position
displayed moderate to low activities. For example, new
analogues 10 and 11 exhibited a similar or significantly higher
potency than niclosamide. However, compound 4 with a
methylated terminal amino group in 4-O-piperidinyl moiety
displayed 10-fold loss of antiproliferative activity in comparison
with analogue 10. The same trend of SAR was also found for
compounds 5−8, in which the dialkylsubstitution at the
terminal amino group resulted in significant loss of activities,
while O-ethylpiperazinyl derivative 9 in contrast with 8
regained antiproliferative activity with IC50 values of 12.4 and
8.7 μM against two breast cancer cell lines MCF-7 and MDA-
MB-231, respectively. The fluorinated compound 3 that was
initially designed for potential PET imaging studies displayed
no significant effects against the tested cancer cells.
a
Reagents and conditions: (a) 16, Ph3P, DIAD, THF, rt, 58%. (b) Zn
dust, sat. NH4Cl (aq.), MeOH, rt. (c) MeC(O)Cl or MeSO2Cl, Et3N,
CH2Cl2, rt. (d) TFA, CH2Cl2, 0 °C to rt, 31−66% (three steps).
of 18 with methanesulfonyl chloride or acetyl chloride followed
by removal of the Boc group afforded the desired products 19
and 20, respectively. As shown in Table 1, replacement of the
nitro group of compound 10 with methanesulfonamide (19) or
acetamide (20) resulted in a significant loss of the
antiproliferative activity, indicating that the nitro moiety is
fairly important for potency.
From the in vitro screening of all tested compounds,
analogues 10 (HJC0125) and 11 (HJC0152) stood out with an
attractive anticancer profile and were therefore subjected to
further biological evaluation. To study the effects of these two
compounds on cell growth, cellular morphological changes
were examined in MDA-MB-231 breast cancer cells treated
with compounds 10, 11, or niclosamide for 48 h, under light
microscopy. Like niclosamide, both compounds 10 and 11
significantly inhibited cell proliferation and induced apoptosis
accompanying cellular morphological changes at concentrations
of 1, 5, and 10 μM, respectively (Supporting Information).
Further modifications of nitro group in compound 10, one of
the most potent analogues identified from our first SAR
exploration, were also investigated. As outlined in Scheme 2,
reduction of the nitro group of the intermediate 17 with zinc
dust provided the corresponding amine 18. Further treatment
B
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Because one of major goals for our discovery effort was to
identify compounds with improved water solubility and oral
bioavailability, aqueous solubility of the new analogues 10 and
11 was determined by an HPLC method.26 As expected,
introduction of the O-alkylamino-tethered moiety not only
enhanced anticancer activity but also significantly increased the
aqueous solubility. Both compounds 10 and 11 (in the form of
HCl salt) demonstrated excellent water solubility, with a
saturated concentration of 248 and 762 μg/mL, respectively
(Figure 1). The O-ethylamino derivative 11 showed the most
Figure 1. Aqueous solubility of novel niclosamide analogues.
Compounds 10 (HJC0125) and 11 (HJC0152) showed significantly
improved solubility as compared with niclosamide.
favorable solubility, which is about 3300-fold improvement in
comparison with that of niclosamide (0.23 μg/mL).27 The
significant improvement of the aqueous solubility is expected to
facilitate these compounds to be more orally bioavailable and
efficacious in vivo than niclosamide, which is substantially water
insoluble.
Figure 2. (A) HJC0152 inhibited the STAT3-mediated luciferase
reporter activity in MDA-MB-231 cells. (B) Proliferation of MDA-
MB-231 cells treated with HJC0152 and niclosamide for 24 h.
Previous studies have demonstrated that niclosamide is a
potential inhibitor of STAT3.19 To determine whether the new
derivatives act as potent small-molecule inhibitors of STAT3
activation, we measured the effect of niclosamide and
compound 11 on promoter activity using the cell-based
transient transfection and dual luciferase reporter assays.
MDA-MB-231 cells were pretreated with niclosamide or 11
at different concentrations for 24 h. The STAT3 promoter
activity in MDA-MB-231 cells was determined after transient
transfecting with pSTAT3-Luc vector. As shown in Figure 2A,
treatment with 10 μM compound 11 decreased the STAT3
promoter activity in MDA-MB-231 cells by approximately 32%,
and increasing the dose of compound 11 to 20 μM further
decreased STAT3 promoter activity by 62% as compared with
control. Similarly, STAT3 promoter activity in MDA-MB-231
cells was suppressed after treatment with 20 μM niclosamide,
while proliferation of MDA-MB-231 cells (MTT assay)19
showed comparable to slightly more potent reduction (Figure
2B). These results demonstrate that compound 11 inhibits
STAT3 promoter activity in MDA-MB-231 cells in a dose-
dependent manner and has a similar effect as niclosamide.
To further investigate the inhibitory activity of compound 11
against the STAT3 pathway, we examined STAT3 phosphor-
ylation and expression of the known STAT3 target genes in
MDA-MB-231 cell line. The cells were treated with different
doses of compound 11 for 24 h, and levels of total STAT3 and
phosphorylated STAT3 at Tyr-705 were then examined by
Western blot. As shown in Figure 3, total STAT3 was reduced
after treatment with 11 or niclosamide, suggesting that these
compounds may alter transcription and/or translation of
STAT3. Similarly, phosphorylated STAT3 (p-STAT3) at Tyr-
705 is suppressed by 11 or niclosamide, suggesting that
compound 11 has a comparable potency in downregulating
STAT3 protein production and phosphorylation at the Tyr-705
site. Blocking STAT3 signaling in many different tumor cells
leads to induce growth arrest and apoptosis.28−30 We observed
that compound 11 induced cleaved caspase-3 and down-
regulated cyclin D1 in MDA-MB-231 cells. These results
suggest that compound 11 inhibits cell cycle progression and
promotes apoptosis. We further confirmed these results using
annexin V-based measurement using flow cytometry. As
depicted in Figure S2 in the Supporting Information,
compound 11 and niclosamide activated apoptosis in MDA-
MB-231 breast cancer cells in a dose-dependent manner. Other
than targeting STAT3, niclosamide has also been found to
inhibit NF-kB and Wnt/β-catenin signaling.19,31,32 Currently,
we are investigating the mechanisms of how compound 11
regulates STAT3 upstream targets and related signaling
pathways and transcriptional/translational regulation.
Furthermore, compound 11 was further evaluated for its
antitumor activity in inhibition of tumor growth in the MDA-
MB-231 xenograft model. As expected, mice treated with 7.5
mg/kg of compound 11 via ip showed a better effect in
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Figure 3. (A) Western blot analysis of biochemical markers for
apoptosis induction and inhibition of STAT3 activity by HJC0152 in
the MDA-MB-231 cell line. (B) Densitometric analysis of three
independent experiments for the expression level of total STAT3,
phospho-STAT3, cyclin D1, and cleaved caspase 3. * represents p <
0.05, and ** represents p < 0.01.
inhibiting tumor growth as compared to the mice treated with
12.5 mg/kg niclosamide (Figure 4A). Similarly, we treated the
MDA-MB-231 xenograft mice with oral administration of
compound 11 and found that the growth of xenograft tumors
in mice was significantly reduced by compound 11 at a dose of
25 mg/kg and even more efficacious than niclosamide at 75
mg/kg (Figure 4B). This observation might be attributed to the
superior solubility of compound 11, which might have resulted
in an improved oral bioavailability33−37 and, consequently, an
enhanced suppression of tumor growth in mice. It is also
noteworthy that compound 11 did not show significant signs of
toxicity at a dose of 75 mg/kg. These results have demonstrated
that compound 11 (HJC0152) is a promising anticancer drug
candidate with excellent aqueous solubility and have significant
potential to be developed as an orally bioavailable anticancer
agent.
In summary, a series of novel O-alkylamino tethered
derivatives of niclosamide have been designed, synthesized,
and biologically evaluated in this work. New analogues 10 and
11 were identified to exhibit a similar or significantly higher
potency than niclosamide against human breast and pancreatic
cancer cells. Both compounds 10 and 11 have demonstrated a
superior aqueous solubility, especially the compound 11, which
has about 3300-fold improvement in water solubility in
comparison with niclosamide. In MDA-MB-231 cells, com-
Figure 4. In vivo efficacy of compound 11 (HJC0152) in inhibiting
growth of xenograft tumors (breast cancer MDA-MB-231) in mice.
(A) ip and (B) po.
pound 11 inhibited STAT3 promoter activity, increased the
expression of active caspase-3, inhibited cell cycle progression,
and promoted apoptosis. In nude mice bearing breast tumor
xenografts, compound 11 significantly suppressed MDA-MB-
231 xenograft tumor growth in vivo (both ip and po). These
results suggest that compound 11 (HJC0152) with remarkably
improved aqueous solubility may have great potential to be
developed as an orally bioavailable agent for cancer therapy.
ASSOCIATED CONTENT
■
S
* Supporting Information
Supplemental data, spectroscopic data with methods of
synthesis for all compounds, and methods of biological
evaluation. This material is available free of charge via the
Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: 713-834-6357. Fax: 713-834-6350. E-mail: QShen@
mdanderson.org (Q.S.). Tel: 409-772-9748. Fax: 409-772-9818.
E-mail: jizhou@utmb.edu (J.Z.).
■
Author Contributions
§These authors contribute equally to this work.
Funding
This work was supported by Grants P50 CA097007,
P30DA028821, and R21MH093844 (J.Z.) from the National
Institute of Health, R. A. Welch Foundation Chemistry and
Biology Collaborative Grant from Gulf Coast Consortia
(GCC), John Sealy Memorial Endowment Fund (J.Z.), the
Duncan Family Institute (DFI) Seed Funding Program, and
startup funds from MD Anderson Cancer Center (Q.S.).
Notes
The authors declare no competing financial interest.
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