Discovery of degradable niclosamide derivatives able to specially inhibit small cell lung cancer (SCLC)

Article abstract of DOI:10.1016/j.bioorg.2020.104574

Small cell lung cancer (SCLC) is exceedingly tough to treat and easy to develop resistance upon long use of the first-line drug carboplatin or radiotherapy. Novel medicines effective and specific against SCLC are greatly needed. Herein, we focused on the discovery of such a medicine by exploring a drug niclosamide with repurposing strategy. Initial screening efforts revealed that niclosamide, an anthelmintic drug, possessed the in vitro anticancer activity and an obvious sensitivity towards SCLC. This observation inspired the evaluation for two different kinds of niclosamide derivatives. 2 with a degradable ester as a linker exhibited the comparable activity but slightly inferior selectivity to SCLC, by contrast, the cytotoxicities of 4 and 5 with non-degradable ether linkages completely disappeared, clearly validating the importance of 2-free hydroxyl group or 2-hydroxyl group released in the antitumor activity. Mechanism study unfolded that, similar to niclosamide, 2 inhibited growth of cancer cells via p 53 activation and subsequent underwent cytochrome c dependent apoptosis. Further structural modification to afford phosphate sodium 8 with significantly enhanced aqueous solubility (22.1 mg/mL) and a good selectivity towards SCLC demonstrated more promising druggability profiles. Accordingly, niclosamide as an attractive lead hold a huge potential for developing targeted anti-SCLC drugs.

Full text of DOI:10.1016/j.bioorg.2020.104574

Bioorganic Chemistry 107 (2021) 104574
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Discovery of degradable niclosamide derivatives able to specially inhibit
small cell lung cancer (SCLC)
a,
b
c
f
d,e,*
XingGang He , MaoLin Li , WenChong Ye , Wen Zhou
a
Shool of Pharmacy, Shanghai Jiaotong University, Shanghai, 800 Dongchuan Rd, 200240 Shanghai, China
Beijing XinDe RunXing Technology Co. Ltd., Beijing, China
b
c
School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, 510120 Guangdong, China
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 200241 Shanghai, China
Key Laboratory of Veterinary Chemical Drugs and Pharmaceutics, Ministry of Agriculture and Rural Affairs, Shanghai Veterinary Research Institute, Chinese Academy
d
e
of Agricultural Sciences, Shanghai 200241, China
f
School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, E. 232, University Town, Waihuan Rd, Panyu, Guangzhou 510006, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Small cell lung cancer (SCLC) is exceedingly tough to treat and easy to develop resistance upon long use of the
first-line drug carboplatin or radiotherapy. Novel medicines effective and specific against SCLC are greatly
needed. Herein, we focused on the discovery of such a medicine by exploring a drug niclosamide with repur-
posing strategy. Initial screening efforts revealed that niclosamide, an anthelmintic drug, possessed the in vitro
anticancer activity and an obvious sensitivity towards SCLC. This observation inspired the evaluation for two
different kinds of niclosamide derivatives. 2 with a degradable ester as a linker exhibited the comparable activity
but slightly inferior selectivity to SCLC, by contrast, the cytotoxicities of 4 and 5 with non-degradable ether
linkages completely disappeared, clearly validating the importance of 2-free hydroxyl group or 2-hydroxyl group
released in the antitumor activity. Mechanism study unfolded that, similar to niclosamide, 2 inhibited growth of
cancer cells via p 53 activation and subsequent underwent cytochrome c dependent apoptosis. Further structural
modification to afford phosphate sodium 8 with significantly enhanced aqueous solubility (22.1 mg/mL) and a
good selectivity towards SCLC demonstrated more promising druggability profiles. Accordingly, niclosamide as
an attractive lead hold a huge potential for developing targeted anti-SCLC drugs.
Discovery
Niclosamide
Water-solubility
Anti-SCLC
Selectivity
1
. Introduction
Small cell lung cancer (SCLC), a heterogeneous and genetically
with an extremely poor prognosis, demanding participant of the second-
line chemotherapy [4]. Toptecan, clophosphamide, doxorubincin and
vincristine were reported as the second-line SCLC’s drugs [5]. Addi-
tionally, a plethora of targeted agents alone or in combination with
conventional chemotherapy in the treatment of SCLC were widely
studied. However, various targeted tyrosine kinase inhibitors (TKIs)
including EGFR TKIs, BCR-ABL TKIs, as well as targeted mTOR in-
hibitors, had failed to demonstrate a survival advantage in SCLC [2,6].
More disappointingly, clinical antiangiogenic agents, such as bev-
acizumab, thalidomide and sorafenib, had no improvements on overall
survival (OS) [7]. Therefore, the limitations of current chemotherapy
drugs necessitated the continuous exploration of novel chemothera-
peutic agents.
complex disease, is an aggressive neuroendocrine lung neoplasm, ac-
counting for approximately 15–20% of all lung cancers [1]. It comprises
limited stage (LS) disease with tumor confined to one hemithorax, and
extensive stage (ES) disease with metastasis beyond one hemithorax [2].
Clinically, concurrent chemo-radiotherapy is a main remedy for LS
disease, while chemotherapy alone is used to treat ES disease [2]. In
comparison to overall 5-year survival rates of 16% for non-small cell
lung cancer (NSCLC), the best currently available therapies for patients
suffering from SCLC achieve only 6%, [3] emphasizing the complexity of
SCLC and the urgent need for developing anti-SCLC drugs.
Platinum-containing agents always served as the first-line treatment
drugs for SCLC. Long exposure to carboplatin posed a “refractory” SCLC
Niclosamide (5-chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenza
mide, 1) was an FDA-approved salicylanilide derivative to treat
*
Corresponding author at”: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 200241 Shanghai, China.
E-mail address: zhouwen60@126.com (W. Zhou).
https://doi.org/10.1016/j.bioorg.2020.104574
Received 19 October 2020; Received in revised form 16 December 2020; Accepted 17 December 2020
Available online 28 December 2020
0
045-2068/© 2020 Elsevier Inc. All rights reserved.

X. He et al.
Bioorganic Chemistry 107 (2021) 104574
tapeworm infections [8]. With an increase in drug repurposing initia-
tives, niclosamide had emerged as a true lead in the screening campaign
against a large cohort of human cancers [9–13]. Growth inhibition of
various cancer cells and reversal of drug resistance [12] were results of
niclosamide’s ability to regulate a selection of signaling pathways
2, 4 and 5 (Scheme 1), commercially available niclosamide 1 as the
starting material was converted into acetate 2 in the presence of acetyl
chloride[25] or acetic anhydride, [26] but the yield was unsatisfactory
due to the generation of diacetate 3. A modified procedure for synthe-
sizing 2 was conducted that niclosamide was acylated with excessive
◦
[
[
14–18] and to induce mitochondrial-dependent uncoupling apoptosis
acetic anhydride in anhydrous DMF at 0 C, and only little diacetate 3
19]. Niclosamide eliminated radio-resistance in the lung cancer cells
was observed. Ethers 4 and 5 were prepared by condensation of 2-hy-
droxyl group of niclosamide with the corresponding brominated al-
kanes [27]. As illustrated in Scheme 1, in the presence of potassium
carbonate, potassium iodide and tetrabutyl ammonium bromide
(TBAB), etherification of niclosamide with 1-brompentane and iso-
amylbromide afforded ethers 4 and 5 in 35.4% and 36.6% yields,
respectively.
and the corresponding xenograft tumors via the inhibition of signal
transducer and activator of transcription 3 (STAT3) and nuclear locali-
zation [12]. The similar result was observed that niclosamide exhibited
synergistic effects on cisplatin-resistant lung cancer cells [20–22].
Recently, mitochondrial-dependent uncoupling apoptosis imposed by
niclosamide rendered an unexpected metabolic vulnerability to selec-
tively killing p53-defective cancer cells, [19] suggesting that p53 was
regarded as a new potential biomark for precision treatment of niclo-
samide and its derivatives. To be taken together, these observed anti-
cancer activities and resistance reversal supported the fact that
nicosamide was an attractive anticancer therapeutic agent implicated in
the inhibition of tumor growth and elimination of drug resistance.
To solve the issue of niclosamide’s poor aqueous solubility and its
resulting low bioavailability, five kinds of widely accepted strategies
towards improvements on drug’s water solubility listed in Scheme 2
were studied. Diethyl 6 was obtained by condensation of niclosamide
3
with diethyl phospite followed by hydrolysis with Me SiBr to provide
phosphate 7 in a high yield, and subsequent treatment of 7 with sodium
hydroxide in anhydrous methanol yielded disodium 8 [28,29]. Exposure
to diisopropylethylamine (DIPEA) and dimethylaminopyridine (DMAP),
incorporation of commercially available tertbutyl-4-aminopiperidine-1-
carboxylate hydrochloride to niclosamide was activated by triphosgene
to produce construct 9, which was further converted into bromide 10
with 33.0% HBr-AcOH in a 72.0% yield [30]. In the presence of dicy-
clohexylcarbodiimide (DCC) and DMAP, tertbutyl (2-hydroxyethyl)-
carbamate 11 prepared previously, [31] was coupled with niclosamide
to afford construct 12, and subsequent deprotection with trifluoroacetic
acid (TFA) in DCM produced amine 13. The similar operation as 10 was
introduced to synthesize bromide 14. Direct reaction of niclosamide
[
23,24]
Although niclosamide hold a great potential in inhibiting the sensi-
tive/resistant NSCLC[14–19], little attention was paid in the treatment
of SCLC. As reported, [15] underlying side effects and very poor water
solubility largely compromised the development of niclosamide as an
antitumor drug. It had been demonstrated that niclosamide exhibited
many drug repurposes, [15] hinting other unpredictable bioactivities
probably leading to severe side effects. Besides, extremely poor aqueous
solubility of niclosamide was only amendable to treating tapeworm in-
fections in the gut, but seriously impeded the possibility of developing
an antitumor drug. In this study, we proposed a new paradigm for tar-
geting SCLC according to niclosamide’s molecular structure in the
anticipation of potentially maintaining the antitumor activity and
selectivity but improving water solubility. Attachment of three typical
substituents to 2-hydroxyl group of niclosamide, bearing an ester or an
ether as a linkage, was used to explore a potential modification direc-
tion, and then their in vitro and in vivo antitumor activities towards SCLC
were evaluated, accompanying with the explanation for a possible
mechanism, and ultimately various water-soluble niclosamide de-
rivatives with a good selectivity toward SCLC were developed by further
structural modifications.
◦
with chlorosulfonyl isocyanate yielded sulfanilamide 15 at 50 C [32].
Coupling of 2,3,4,6-tetra-O-acetyl- -D-glucopyranosyl bromide 16 with
α
niclosamide generated acetyl-protected 17 with a yield of 56.2%, [33]
and deacetylation of 17 with a catalytic amount of sodium methoxide in
+
anhydrous methanol followed by acidification with Dowex 50 (H )
resins afforded glucose-coupled 18, [34] with the yield demonstrating
absolutely advantages over the reported synthetic method. [35]
2.2. Biological evaluation
2
. Results and discussion
The in vitro antitumor activities of all the newly synthesized niclo-
samide derivatives were assessed in comparison with a first-line drug
carboplatin and the lead niclosamide 1 against three cancer cell lines
including NSCLC (A549), SCLC (NCI-H446), human T lymphoblasts cell
line (Jurkat) and one normal cell line (human bronchial epithelial cell,
HBE) (Table 1). Carboplatin, a first-line therapy for SCLC, was used a
2
.1. Chemistry
In the synthesis of three typically modified niclosamide derivatives
Scheme 1. Reagents and Conditions: a). Ac2O, DMAP, DMF, 0 oC, yield, 88.2% for 2, 3.3% for 3; b).K2CO3, KI, (Bu)4NBr, 1-brompentane for 4, yield, 35.4%,
isoamyl bromide for 5, yield, 36.6%.
2

X. He et al.
Bioorganic Chemistry 107 (2021) 104574
Scheme 2. Reagents and Conditions: a). i, diethyl phosphite, DIPEA, DMAP, ii, CCl4, DMF, 0 oC, 96.2%; b) Me3SiBr, DCM, 0 oC, yield, 99.6%; c). NaOH, CH3OH,
yield, 86.5%; d). i, tertbutyl-4-aminopiperidine-1-carboxylate hydrochloride, DIPEA, ii, triphosgene, 0 oC, 21.5%; e). 33% HBr-AcOH, DCM, 72.0%; f). tertbutyl(2-
hydroxyethyl)-carbamate 11, DCC, DMAP, yield, 57.0%; g) TFA, DCM, yield, 81.0%; i) chlorosulfonyl isocyanate, 50 oC, yield, 76.0%; h)0.2,3,4,6-tetra-O-acetyl-
α
-D-
glucopyranosyl bromide (16), TBAB, Cs2CO3, yield, 56.2%; j) NaOMe, anhydrous MeOH, yield, 82.7%.
with pentyl (3) and isoamyl (4), respectively, the cytotoxicities against
all the tested cancer cell lines sharply disappeared (IC50 > 100 µM). This
obvious difference
Table 1
The in vitro antitumor activities of niclosamide derivatives against three cancer
cell lines (A549, NCI-H446, Jurkat) and one normal cell line (HBE).
was possibly ascribed to the linkage modes: 2 with a degradable ester
was able to release the parent compound niclosamide in the presence of
a certain esterase, while 4 and 5 bearing a non-degradable ether fail.
From these in vitro observations, niclosamide indeed hold a great
sensitivity toward SCLC and free 2-hydroxyl was an essential pharma-
cophore to the antitumor activities, obviously implying the importance
of 2-OH released, or not, in the antitumor activity.
a
Compds.
no.
R
IC50 (µM)
c
A549
NCI-
Jurkat
HBE
SI
H446
1
2
4
5
H
7.70
4.13
0.96
1.8
6.42
8.0
4.5
/
COCH
3
0.92
1.5
5.76
b
(CH
(CH
2
2
3
)
)
)
4
CH
CH
3
>100
>100
>100
>100
>100
>100
>100
2
2
>100
/
To confirm the in vivo efficacy of 2 whether was in accordance with in
vitro observation, BALB/c mice suffering from SCLC NCI-H69 was used
as an animal evaluation model. Twenty-five female mice tested were
randomly divided into five groups, and each group had five mice. The
first-line drug carboplatin and PBS were served as a positive control and
a negative one, respectively. As shown in Table 2, although, compared
with the positive compound carboplatin (47.3%), 2 administrated with
(
CH
Carboplatin
22.2
37.40
44.66
>100
0.59
a
indicates the average value of the three independent experiments
b
c
indicates that more > 100 µM is ineffective, SI means selectivity index
indicates that SI is a ratio of IC50 (A549)/IC50 (NCI-H446).
positive compound [4]. An IC50 value was the expression for the in vitro
antitumor activity, and it meant the half-maximal inhibitory concen-
tration of a compound to inhibit cell growth.
5
0 mg/kg/d intragastrically exhibited a slightly inferior antitumor ac-
tivity with a TGI of 35.6%, no overt toxicity was apparently observed.
Moreover, at the same dosing (50 mg/kg/d), the antitumor potency of 2
was comparable to that of the lead niclosamide 1 in inhibiting growth of
To ascertain whether 2-hydroxyl group of niclosamide as a potential
modification direction or not, three typical niclosamide derivatives (2, 4
and 5) were explored using a MTT-based cell assay. Noticeably,
although acetate 2 was reported be effective in inhibiting plentiful of
Table 2
,
cancer cells [36,37] the involvement of its specificity toward SCLC and
In vivo antitumor activity of niclosamide derivative 2 and carboplatin.
its selectivity between SCLC and NSCLC remained blank. As listed in
Table 1, among three cancer cell lines and one normal cell line, the lead
compound niclosamide displayed obvious selectivity toward SCLC cells
NCI-H446 (IC50 = 0.96 µM) with a SI (selectivity index) of 8.0, indi-
cating the great potential of niclosamide as a true lead in developing
anti-SCLC drugs with high specificity. However, the first-line drug car-
boplatin exhibited a preference for A549 cells (SI = 0.45). Modification
on 2-OH of niclosamide with acetyl (2), had no influence on anti-SCLC,
whereas the cytotoxicity against A549 (IC50 = 4.13 µM) increased by
twofold in comparison with niclosamide (IC50 = 7.70 µM), giving rise to
a certain decrease in the SI of 2 (SI = 4.5). When acetyl was replaced
a
Group
Name
Administration
route
Size of
Dosage
(mg/kg/
d)
Animal
TGI
(%)
animals
numbers left
on Day 29
N.C
i.g.
i.g.
i.g.
i.g.
i.p.
5
5
5
5
5
/
5
5
5
5
4
0
Carboplatin
6
47.3
35.6
32.4
/
2
1
2
50
50
50
i.g. means intragastrically; i.p. means intraperitoneally; TGI means tumor
growth inhibition, which is equal to a ratio of weight of treatment group to
weight of control group.
3

X. He et al.
Bioorganic Chemistry 107 (2021) 104574
tumor. Anatomically, most of 2 or 1 was accumulated in the gut as solid
powders. This was a good explanation for high dosing (50 mg/ kg/d) but
unintended effect. When administrated intraperitoneally, one mouse
died due to excessive insoluble solid 2 accumulated in all the organs of
abdomen. Noticeably, no efficacy was found when treated with com-
pound 4 or 5 at the same dose (data not offered). Collectively, 2
displayed the in vivo antitumor activity in line with its in vitro one,
but it was severely compromised by poor aqueous solubility, even
leading to unexpected toxicity when administrated intraperitoneally.
Mutation in p53 prevailed across a variety of human cancers.
Niclosamide was showed to trigger optimal protective p53 activation
while inducing cytotoxicity, as a result, growth of p53-deficient cells and
of p53 mutant patient-derived ovarian xenografts was significantly
impaired[19] To determine whether 2 induced p53 activation and
exerted the anti-SCLC activity or not, the expression of p53 protein in
SCLC (NCI-H446) as a tested cell model was explored. As demonstrated
in Fig. 1, NCI-H446 cells did not belong to p53-deficient cells, but a
significant increase of p53 protein was observed when to be fully
exposed to 2, possibly indicating the direct association of p53 activation
and antitumor activity. Moreover, the inducing effect on p53 in NCI-
H446 was comparable to that of the template compound niclosamide
release by a certain enzyme at a targeted location.
In search of the optimal moiety possessing high hydrophilia or
contributing to the formation of organic salt with base or acid, five
widely accepted strategies were selected to improve water solubility of
niclosamide. According to the procedure for measuring aqueous solu-
bility reported previously, [44] the data were summarized in Table 3.
Niclosamide and its acetyl derivative 2 were very poor soluble in
aqueous solution at pH = 7.4 (0.06 mg/mL for 1 and 0.05 mg/mL for 2).
Niclosamide phosphate was beneficial to an increase in water solubility,
but no data was available for the antitumor activity [45]. As expected,
the incorporation of phosphate (7, 7.2 mg/mL) and disodium phosphate
(8, 22.1 mg/mL) resulted in 120-fold and 368-fold increases in aqueous
solubility, respectively. In contrast, 15 carrying sulfanilamide, 14 with
glycine and 18 endowed with glucose exhibited limited enhancements
in water solubility compared with niclosame 1. Unfortunately, aqueous
solubility of 10 can be unmeasurable due to its instability exposed to
methanol contained an eluent in HPLC analysis. Taken together, among
five strategies we used, niclosamide modified with phosphate was the
optimal option to solve aqueous solubility.
Since all the newly synthesized compounds displayed by far better
water solubility over the lead niclosamide and acetyl derivative 2,
release of niclosamide was crucial in the antitumor activities and a
selectivity towards SCLC. 2 was selected as a positive control, and A549
cells (NSCLC) and NCI-H 446 cells (SCLC) were served as cell evaluation
models. Seen in Fig. 2, compounds 8 and 10 exhibited the comparable
antitumor activities to 2, possibly suggesting similar release of the lead
niclosamide from 8 and 10 as 2. The antitumor activity of 14 was
inferior to those of 8 and 10, which was possibly due to the difference of
releasing ability. Moreover, three derivatives were sensitive towards
NCI-H446 with the non-differential selectivity (SI = 2.90 for 8, 2.3 for
10, 2.7 for 14). By contrast, A549 cells and NCI-H 446 cells were
insensitive to 15 with sulfanilamide and 18 carrying glucose, empha-
sizing that sulfamide and glycosidic bond were tolerant to SCLC and
NSCLC. The difference in the antitumor activities of these tested com-
pounds possibly originated from the releasing ability. Therefore, from
the angels of antitumor activity and water solubility, 8 with disodium
phosphate exhibited better physicochemical property and a good
selectivity towards NCI-H 446 cells, possibly contributing to potential
bioavailability as an anti-SCLC drug.
1
. As reported [19], 2-free hydroxyl group of niclosamide was an
essential pharmacophore in sensitizing p53 knockout cells. A niclosa-
mide derivative with a methoxy group (–OCH ) instead of a hydroxyl
3
one (2-OH) had no effect on growth of either wildtype or p53-deficient
cells even at high concentrations [19]. This was a reasonable explana-
tion for our results that compounds 4 and 5 failed to release 2-OH and
sharply damaged the antitumor activities (Table 1). Moreover, p53
activation of 2 was probably due to cleavage of an acetyl group from 2.
In addition, we further extended our analysis to cytochrome c (Cyt c)
associated with cell apoptosis and heat shock protein (Hsp90) respon-
sible for controlling distinct client proteins via protein folding [19,38]. A
significant increase in Cyt c release in response to 2 and 1 was observed
(
Fig. 1), but leaving Hsp90 minimally affected. This supported the
possibility that 2 inhibited growth of NCI-H446 via p53 activation and
subsequent underwent cytochrome c dependent cell apoptosis, without
participation of molecular chaperone Hsp90.
From the in vivo biological assessment of 2, the hypothesis was
further supported that the low aqueous solubility placed severe limits on
its antitumor efficacy. Poor aqueous solubility and the resulting low
bioavailability indeed posed a huge obstacle to developing anticancer
medications from niclosamide as a lead. To exert their bioactivities of
niclosamide and its derivatives, it was very urgent to facilitate the
dissolution. Several approaches to increases in the water solubility of
niclosamide had been reported, including crystal particle size milled to
nanoscale, [39] co-crystallization with safe materials, [40] complexa-
tion with O-phosphorylated calixarene, [41] prodrug formed by incor-
poration of a hydrophilic functional group [42] and solublization via
attachment of octenylsuccinate hydroxypropyl phytoglycogen. [43] Of
note, prodrug design hold attractive advantages over other strategies,
including decreased toxicity, enhanced water solubility, and site-specific
3. Conclusion
In this study, the antitumor properties of the anti-helminthic drug
niclosamide sensitive to SCLC encouraged synthesis and evaluation of
niclosamide-derived agents. The crucial observation from the initial in
vitro screen highlighted the importance of 2-hydroxyl group of niclosa-
mide in the antitumor activity. 2-hydroxyl group capped with alkane
chains including pentyl 4 and isopentyl 5 resulted in the disappearance
of the antitumor activities, while compound 2 with an acetyl group
remained the potent antitumor activity at a nanomolar concentration
and displayed a good selectivity towards SCLC, implying the cleavage of
Fig. 1. Effects of niclosamide (1) and its derivative (2) on several proteins of SCLC (NCI-H446). A. p53, Cyt c and Hsp90 expressions measured by western blotting
analysis. B. Quantification of p53, Cyt c and Hsp90 proteins (Error bars in B indicate SD of the three independent experiments. The significance of difference between
the treated group and the control one was analyzed by Student’s t test, *p < 0.05, ** p < 0.01).
4

X. He et al.
Bioorganic Chemistry 107 (2021) 104574
Table 3
Aqueous solubility of niclosamide and its derivatives at pH 7.4.
a,b
a,b
Compds.
Strategies towards water solubility
Solubility (mg/mL)
Compds.
Strategies towards Water solubility
Solubility (mg/mL)
1
7
0.06
7.2
2
0.05
22.1
1.2
Phosphate
8
Disodium phosphate
Primary amine salt
Glycosylation
c
10
5
Piperidine salt
Sulfanilamide
/
14
18
1
0.2
1.4
a
b
c
solution (pH = 7.4 and 37 ◦C)
Aqueous solubility was measured in mg/mL of a compound tested in 0.1 M Na
means the mean value of determinations in triplicate.
2 4
HPO
indicates unmeasurable due to instability to methanol contained as an eluent.
Fig. 2. Cytotoxicities of all the newly-synthesized water-soluble niclosamide derivatives against NSCLC A549 (A) and SCLC NCI-H446 (B).
ester 2 to release 2-hydroxyl group, but failure to ethers 4 and 5 in the
cell culture medium. In vivo antitumor activity of 2 further supported the
possibility that 2-hydroxyl group was an essential pharmacophore.
Mechanism study showed that 2 induced Cyt c release dependent cell
apoptosis via p53 activation. Of five strategies to improve niclosamide’s
aqueous solubility, structural modification with phosphate to afford 8
exhibited better water solubility, comparable anti-SCLC activity to 2 and
an obvious preference in the inhibition of SCLC. The failure to inhibit in
vitro antitumor activity of other derivatives suggested release of 2-
hyroxyl group was a promising characteristic of novel niclosamide an-
alogues. Identification of the molecular target of these compounds
would provide a guiding template for structural optimization in the
development of specific SCLC inhibitors.
4.1.1. Synthesis of 4-chloro-2-((2-chloro-4-nitrophenyl)carbamoyl)phenyl
acetate (2) and 2-(acetyl(2-chloro-4-nitrophenyl)carbamoyl)-4-
chlorophenyl acetate (3)
To a solution of niclosamide 1 (0.13 g, 0.4 mmoL) and pyridine
(0.036 mL, 0.44 mmoL) dissolved in dry THF (5 mL) under an argon
atmosphere at room temperature was added acetyl chloride (0.034 g,
0.44 mmoL), and a white precipitate was formed over 3 hrs. The reaction
mixture was neutralized with diluted acid, and then extracted with ethyl
acetate (EtOAc, 100 mL). The organic layer was washed with brine once,
2 4
and dried over anhydrous Na SO , and filtered, and then concentrated
under reduced pressure to produce the crude residue, which was further
chromatographed over silica gel with a mixture of EtOAc/petroleum
ether (V/V = 1/20) as an eluent to give 38.4 mg of the desired 2 and
8
4.9 mg of by-product 3, respectively.
4
. Experiential section
Compound 2: Yield, 25.2%, 1H NMR (400 MHz, DMSO) δ 10.48 (s,
H), 8.41 (s, 1H), 8.29 (d, 1H, J = 8.0 Hz), 8.09 (d, 1H, J = 8.0 Hz), 7.85
1
4
.1. Chemistry
(d, 1H, J = 4.0 Hz), 7.71 (d, 1H, J = 4.0 Hz), 7.37 (d, 1H, J = 8.0 Hz),
2
.36 (s, 3H). 13C NMR (100 MHz, DMSO) δ 169.2, 163.6, 147.4, 145.0,
All chemicals were of reagent grade quality or better, which were
141.2, 132.5, 130.5, 130.2, 129.7, 127.7, 126.5, 126.0, 125.5, 123.5,
-
-
purchased from commercial suppliers and used without further purifi-
cation. Solvents were used as received or dried over molecular sieves.
Column chromatography was performed on silica gel (100–200 mesh)
purchased from Qingdao Ocean Chemical Factory. All the reaction
processes were monitored by TLC (HSGF 254) from Yantai Jiangyou
Silica Gel Development Co. LTD (Yantai, China). All the key in-
21.2. TOF-MS, m/z: [Mꢀ H ], Calcd for C15Cl
2
H
9
N
2
O
5
, 366.9894, Found,
366.9885.
1
Compound 3: Yield, 51.6%, H NMR (500 MHz, CDCl
3
) δ 8.35 (d, 1H,
J = 3.5 Hz), 8.11 (dd, 1H, J = 8.5, 3.5 Hz), 7.46 (d, 1H, J = 8.5 Hz), 7.40
(d, 1H, J = 2.5 Hz), 7.37 (dd, 1H, J = 8.5, 3.5 Hz), 7.08 (d, 1H, J = 8.5
Hz), 2.45 (s, 3H), 2.33 (s, 3H). 13C NMR (125 MHz, CDCl
) δ 171.6,
3
1
13
termediates and final products were confirmed by H NMR and
C
168.6, 166.4, 148.0, 146.4, 141.9, 134.3, 132.4, 131.6, 131.3, 129.1,
1
NMR, recorded in a Bruker Avance 400 or 500 ( H at 400 MHz or 500
127.8, 125.7, 124.9, 123.0, 25.9, 20.9.
1
3
MHz, C at 100 MHz or 125 MHz), and chemical shifts were reported in
parts per million using the residual solvent peaks as internal standards
4.1.2. Modified synthetic procedure for compound 2
1
13
(
CDCl
3
= 7.26 ppm for HNMR and 77.16 ppm for CNMR, CD
3
SOCD
3
To a solution of niclosamide (0.65 g, 2.0 mmoL) and DMAP (0.12 g,
1.0 mmoL) dissolved in anhydrous DMF was added acetic anhydride
13
=
2.50 ppm for 1H NMR and 39.52 ppm for C NMR, CD
3
OD = 3.31
ppm for 1H NMR, and 49.00 ppm for 13C NMR). Also, several key in-
termediates and final products were determined by electrospray ioni-
zation high resolution mass spectrometry (ESI-HRMS), recorded on AB
Sciex triple TOF 5600 + system. The purity was>95.0%, and it was
determined with C18-column (250 × 4.6 mm, 5 µm, Agilent Eclipse Plus)
run on Agilent Technologies 1260 infinity II.
(0.5 mL, 4.8 mmoL) dropwise at 0 C. The mixture was stirred at the
◦
same temperature overnight. After finished, the reaction mixture was
diluted with double distilled water (50.0 mL), extracted with EtOAc
(100.0 mL) twice. The combined organic layer was washed with water
2 4
(50.0 mL) and brine (100.0 mL), and dried over anhydrous Na SO , and
then concentrated to afford the crude residue, which was purified by
silica gel-based chromatography with a mixture of petroleum ether and
EtOAc (V/V = 20/1) to provide 0.65 g of 2 as a white solid in an 88.2%
yield.
5

X. He et al.
Bioorganic Chemistry 107 (2021) 104574
4
.1.3. 5-chloro-N-(2-chloro-4-nitrophenyl)-2-(pentyloxy)benzamide (4)
Niclosamide (0.65 g, 2.0 mmoL), KI (0.16 g, 1.0 mmoL), Bu NBr
0.16 g, 0.5 mmoL) and K CO (0.84 g, 12.0 mmoL) were taken up in
,4-dioxane (50.0 mL) in order, and 1-brompentane (1.22 g, 16.0 mmoL)
4.1.7. 4-chloro-2-((2-chloro-4-nitrophenyl)carbamoyl)phenyl phosphate
(8)
4
(
2
3
To 20.0 mL of a stirred methanol solution of 7 (1.2 g, 2.96 mmoL)
was added dropwise saturated sodium hydroxide. When the sediment
appeared, the solution was filtered, and freeze-dried to afford 0.72 g of 8
as a white powder.
1
was added dropwise. The resultant mixture was heated to 85 ℃ and
stirred overnight. After completion of the reaction, 1,4-dioxane was
removed under reduced pressure. The obtained residue was re-dissolved
with dichloromethane (DCM) and washed with water twice. The organic
1
Yield, 54.0%. H NMR (400 MHz, DMSO) δ 8.95 (d, 1H, J = 9.2 Hz),
8.43 (s, 1H), 8.27 (d, 1H, J = 2.8 Hz), 8.13 (dd, 1H, J = 9.2, 2.8 Hz), 7.67
3
1
layer was dried over anhydrous Na
SO
2 4
, filtered and concentrated to
(s, 1H), 7.05 (dd, 1H, J = 4.0, 4.0 Hz), 6.50 (t, 1H, J = 8.0 Hz). P NMR
13
give the crude product, which was purified over silica gel column with
(160 MHz, DMSO) δ ꢀ 4.72. C NMR (100 MHz, DMSO) δ 168.4, 167.8,
147.1, 139.9, 132.2, 128.2, 124.7, 123.4, 122.6, 122.5, 120.6, 119.0,
114.4.
an eluent containing petroleum ether and EtOAc (V/V = 15/1) to pro-
vide 0.25 g of 4 as a white solid.
1
Yield, 35.4%, H NMR (400 MHz, CDCl
3
) δ 10.57 (s, 1H), 8.87 (d, 1H,
J = 8.0 Hz), 8.45 (s, 1H), 8.31 (d, 1H, J = 4.0 Hz), 8.21 (d, 1H, J = 4.0
4.1.8. tertbutyl4-(((4-chloro-2-((2-chloro-4-nitrophenyl)carbamoyl)
phenoxy) carbonyl)amino)piperidine-1-carboxylate (9)
Hz), 8.19 (d, 1H, J = 8.0 Hz), 4.27 (t, 2H, J = 8.0 Hz), 1.88–1.95 (m, 2H),
1
.37–1.40 (m, 4H), 0.88 (s, 3H).
A solution of tertbutyl 4-aminopiperidine-1-carboxylate hydrochlo-
ride (3.0 g, 12.7 mmoL) in DCM (40.0 mL) was fiercely mixed with
saturated sodium bicarbonate solution (30.0 mL). The organic phase was
4
.1.4. 5-chloro-N-(2-chloro-4-nitrophenyl)-2-(isopentyloxy)benzamide
(
5)
4
dried over anhydrous MgSO , and then concentrated under reduced
The synthetic procedure for compound 5 was similar as that of 4,
pressure to afford 2.3 g of 4-aminopiperidine-1-carboxylate as a yellow
◦
only differing in isoamyl bromide as a starting material instead of 1-
oil in a 91.0% yield. Next, a cooled solution (0 C) of triphosgene (3.54 g,
brompentane.
11.96 mmoL) in dry DCM (10.0 mL) was introduced to a dry DCM so-
lution (10.0 mL) of the above yellow oil (2.3 g, 11.5 mmoL) and N,N-
diisopropylethylamine (3.8 mL, 23.0 mmoL) in a dropwise fashion. The
mixture was allowed to warm to r. t. and stirred for 1.0 h, and then
concentrated under diminished pressure to afford the residue without
purification for next reaction. Finally, to a solution of niclosamide (3.76
g, 11.5 mmoL) dissolved in a mixture of dry DCM (10.0 mL) and dry
pyridine (1.0 mL) were added dropwise the above residue and 0.20 g of
4-dimethylaminopyridine. The resulting solution was stirred at r.t.
overnight. After the reaction was over, EtOAc (200.0 mL) was added.
The organic layer was washed with diluted hydrochloric acid (1 M),
1
Yield, 36.7%. H NMR (400 MHz, CDCl
3
) δ 10.54 (s, 1H), 8.88 (d, 1H,
J = 8.0 Hz), 8.45 (s, 1H), 8.30 (d, 1H, J = 4.0 Hz), 8.21 (d, 1H, J = 4.0
Hz), 8.19 (d, 1H, J = 8.0 Hz), 4.30 (t, 2H, J = 8.0 Hz), 1.87 (t, 2H, J =
8
.0 Hz), 1.23 (m, 1H), 0.96 (s, 3H), 0.94 (s, 3H).
4
.1.5. 4-chloro-2-((2-chloro-4-nitrophenyl)carbamoyl)phenyl diethyl
phosphate (6)
To a stirred solution of niclosamide (2.0 g, 6.12 mmoL) dissolved in a
mixture of dry N,N-dimethylformamide (20.0 mL) and carbon tetra-
chloride (3.0 mL) were slowly added N,N-diisopropylethylamine (2.0
◦
mL, 11.5 mmoL) and a catalytic amount of DMAP at 0 C. Subsequently,
2 4
water and brine in order, and dried by anhydrous Na SO , and then
diethyl phosphite (850.0
μ
L, 12.3 mmoL) was added dropwise. The re-
concentrated to afford a yellow solid, which was further purified by
silica gel chromatography with a mixture of EtOAc and petroleum ether
◦
action mixture was stirred for 2.0 hrs at 0 C. After done, the solution
was quenched with 0.50 M K
for another 20 mins. The resultant mixture was extracted with EtOAc
100 mL). The organic layer was washed with brine (30.0 mL), and dried
by anhydrous Na SO , and filtered, and then concentrated under
2
HPO
4
and warmed to r.t., and then stirred
(V/V = 1/10) as an eluent to yield 1.33 g of 9 as an yellow solid.
1
Yield, 21.0%. H NMR (400 MHz, CDCl
3
) δ 8.40 (d, 1H, J = 2.4 Hz),
(
8.24 (dd, 1H, J = 8.0, 2.4 Hz), 8.03 (d, 1H, J = 2.4 Hz), 7.64 (dd, 1H, J =
8.0, 2.4 Hz), 7.50 (d, 1H, J = 8.0 Hz), 7.21 (d, 1H, J = 8.0 Hz), 3.96 (dt,
1H, J = 8.0, 4.0 Hz), 3.62–3.44 (m, 2H), 3.15–3.10 (m, 2H), 1.73–1.55
(m, 2H), 1.41 (s, 9H), 1.38–1.29 (m, 2H).
2
4
reduced pressure to offer the crude product, which was purified by silica
gel-based chromatography with an eluent containing a mixture of EtOAc
and petroleum ether (V/V = 1/2) to yield 2.78 g of 6 as a white powder.
1
Yield, 96.2%. H NMR (500 MHz, CDCl
3
) δ 9.51 (s, 1H), 8.81 (d, 1H,
4.1.9. 4-(((4-chloro-2-((2-chloro-4-nitrophenyl)carbamoyl)phenoxy)
carbonyl) amino) piperidin-1-ium bromide (10)
J = 9.5 Hz), 8.32 (s, 1H), 8.19 (d, 1H, J = 9.5 Hz), 8.03 (s, 1H), 7.51 (d,
3
1
2
H, J = 9.5 Hz), 4.20 (m, 4H), 1.29 (t, 6H, J = 7.5 Hz). P NMR (200
9 (0.3 g, 0.54 mmoL) was dissolved in 33% hydrobromic acid con-
tained in AcOH (3.0 mL) at 0 ℃, and the mixture was stirred at r.t. for
0.5 h. The solvent was directly concentrated under diminished pressure
to yield 0.21 g of 10 as a white solid.
1
3
MHz, CDCl
3
) δ ꢀ 6.06. C NMR (125 MHz, CDCl
3
) δ 162.1, 146.7, 143.4,
40.5, 133.6, 131.9, 131.6, 126.6, 124.9, 123.6, 123.2, 122.7, 121.2,
1
6
5.7, 65.6, 16.2, 16.1.
1
Yield, 72.0%. H NMR (400 MHz, DMSO) δ 8.54 (d, 1H, J = 2.4 Hz),
4
.1.6. 4-chloro-2-((2-chloro-4-nitrophenyl)carbamoyl)phenyl dihydrogen
8.39 (dd, 1H, J = 8.8, 2.4 Hz), 8.35 (s, 1H), 8.14 (s, 1H), 7.98–7.87 (m,
2H), 7.50 (d, 1H, J = 8.8 Hz), 4.08–4.01 (m, 1H), 3.02–2.88 (m, 4H),
1.89–1.75 (m, 2H), 1.54–1.40 (m, 2H).
phosphate (7)
(1.4 g, 3.05 mmoL) was dissolved in dry DCM (7 mL), and trime-
6
◦
thylsilyl bromide (2.4 mL, 18.1 mmoL) was added dropwise at 0 C. The
◦
reaction mixture was stirred at 0 C for 12.0 hrs. When the reaction was
4.1.10. (tert-butoxycarbonyl)glycine (11)
completed, addition of methanol (2.0 mL) was performed. The reaction
mixture was stirred for another 20 mins. The solvent was directly
removed under diminished pressure to afford 1.2 g of 7 as a white
powder.
Glycine (3.0 g, 40.0 mmoL) was dissolved in a mixture of dioxane
(15.0 mL) and water (15.0 mL), and then tri-ethylamine (5.54 mL, 40.0
mmoL) and tert-butyl dicarbonate (8.72 mL, 40.0 mL) were added in
order. The resulting reaction mixture was stirred at r.t. overnight. After
evaporation, the residue was re-dissolved in water, and extracted with
diethyl ether (25.0 mL) to remove some unknown impurities. A resulting
solution was adjusted to pH = 2.0 with diluted hydrochloric acid (2.0
M), and extracted with EtOAc (20.0 mL × 2). The combined organic
layer was washed with water and brine in order, and dried over anhy-
Yield, 95.7%. 1H NMR (500 MHz, DMSO) δ 10.33 (s, 1H), 8.49 (d,
1
7
9
H, J = 9.0 Hz), 8.40 (d, 1H, J = 3.0 Hz), 8.29 (dd, 1H, J = 9.0, 3.0 Hz),
.83 (d, 1H, J = 3.0 Hz), 7.68 (dd, 1H, J = 9.0, 3.0 Hz), 7.55 (d, 1H, J =
.0 Hz,). 31P NMR (200 MHz, DMSO) δ ꢀ 6.11. C NMR (125 MHz,
13
DMSO) δ 162.6, 148.1, 143.4, 140.7, 132.7, 130.1, 128.3, 127.2, 124.8,
1
-
24.7, 123.3, 123.1, 123.0. TOF-MS, m/z: [Mꢀ H] , calcd for
drous Na
2 4
SO , and then concentrated under diminished pressure to
-
13 8 2 2 7
C H Cl N O P , 404.9452, Found, 404.9444.
afford 6.7 g of 11 as a white solid. Yield, 94.5%.
1
H NMR (500 MHz, CDCl
3
) δ 11.81 (d, 1H, J = 7.5 Hz), 5.40–5.14 (m,
6

X. He et al.
Bioorganic Chemistry 107 (2021) 104574
1
4
2
H), 3.91 (t, 1H, J = 6.0 Hz), 3.84 (t, 1H, J = 6.0 Hz), 1.41 (d, 9H, J =
with an eluent containing a mixture of EtOAc and petroleum ether (V/V
.5 Hz). 13C NMR (125 MHz, CDCl
8.2.
) δ 174.6, 157.5, 81.9, 43.4, 28.3,
= 1/6) to yield 4.25 g of 15 as a yellow solid.
3
1
Yield, 76.0%. H NMR (500 MHz, CD
3
OD) δ 8.42 (d, 1H, J = 2.5 Hz),
8
.30–8.19 (m, 2H), 7.72 (dd, 2H, J = 10.5, 8.5 Hz), 7.33 (dd, J = 1H, 9.0,
13
4
.1.11. 4-chloro-2-((2-chloro-4-nitrophenyl)carbamoyl)phenyl
3
1.5 Hz). C NMR (125 MHz, CD OD) δ 153.8, 135.8, 135.0, 131.8,
(
tertbutoxycarbonyl) glycinate (12)
131.2, 130.8, 128.7, 127.8, 126.2, 125.8, 124.4, 123.6, 121.6. TOF-MS,
+
+
Niclosamide (1.86 g, 5.71 mmoL) and 11 (1.0 g, 5.71 mmoL) were
m/z: [M + H ], calcd for C13
H10Cl N O S , 405.9662, found, 405.9666.
2 3 6
dissolved in anhydrous DMF (20.0 mL), and then DMPA (60.0 mg, 0.57
mmoL) and N,N’-dicyclohexylcarbodiimide (1.76 g, 8.57 mmoL) were
added. The reaction solution was stirred at r. t. for 12.0 hrs. After the
reaction was finished, diluted hydrochloric acid (1.0 M) was added
dropwise to neutralize the solution. The reaction mixture was extracted
with EtOAc (200.0 mL), and the organic layer was concentrated under
reduced pressure to afford the crude residue, which was purified by flash
chromatography with an eluent containing a mixture of EtOAc and pe-
4.1.15. (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-bromotetrahydro-2H-
pyran-3,4,5-triyltriacetate (16)
To a solution of penta-acetyl glucopyranose (0.76 g, 1.95 mmoL) in
anhydrous DCM was added 33% hydrobromic acid contained in AcOH
◦
(3.0 mL) at 0 C. The reaction mixture was allowed to heat to r.t. and
stirred for 4.0 hrs. The whole progress was monitored by TLC plate. The
reaction solution was quenched with a mixture of EtOAc (60.0 mL) and
saturated sodium bicarbonate solution (20.0 mL). The organic layer was
washed with saturated sodium bicarbonate solution (20.0 mL × 2),
water (20.0 mL) and brine (20.0 mL) in order, and dried over anhydrous
troleum ether (V/V = 1/8) to yield 1.24 g of 12 as a white solid.
1
Yield, 45.1%. H NMR (500 MHz, CDCl
3
) δ 8.83 (s, 1H), 8.77 (d, 1H,
J = 9.0 Hz), 8.33 (d, 1H, J = 2.5 Hz), 8.20 (dd, 1H, J = 9.0, 2.5 Hz), 7.90
(
d, 1H, J = 2.5 Hz), 7.55 (dd, 1H, J = 9.0, 2.5 Hz), 7.22 (d, 1H, J = 9.0
2 4
Na SO , and filtered. The filter was removed under reduced pressure to
1
3
Hz), 5.09 (t, 1H, J = 6.0 Hz), 4.21 (d, 2H, J = 6.0 Hz), 1.41 (s, 9H).
C
afford 0.73 g of 16 as a white solid.
1
NMR (125 MHz, CDCl
3
) δ 168.8, 162.3, 155.8, 146.4, 143.6, 140.2,
Yield, 91.0%. H NMR (500 MHz, CDCl
3
) δ 6.60 (d, 1H, J = 4.0 Hz),
1
4
33.4, 132.8, 130.3, 128.3, 125.2, 124.9, 123.8, 122.9, 121.0, 80.8,
5.55 (t, 1H, J = 9.5 Hz), 5.15 (t, 1H, J = 9.5 Hz), 4.83 (dd, 1H, J = 9.5,
4.0 Hz), 4.34–4.26 (m, 2H), 4.12 (dd, 1H, J = 12.5, 2.0 Hz), 2.09 (s, 6H),
3.0.
2
.04 (s, 3H), 2.03 (s, 3H). 13C NMR (125 MHz, CDCl
) δ 170.6, 170.0,
3
4
.1.12. 4-chloro-2-((2-chloro-4-nitrophenyl)carbamoyl)phenyl glycinate
169.9, 169.6, 86.7, 72.3, 70.7, 70.3, 67.3, 61.1, 20.9, 20.8, 20.7, 20.6.
(
13)
To a stirred solution of 12 (1.24 g, 2.57 mmoL) in DCM (10.0 mL)
4.1.16. (2R,3R,4S,5R,6S)-2-(acetoxymethyl)-6-(4-chloro-2-((2-chloro-4-
nitrophenyl) carbamoyl) phenoxy)tetrahydro-2H-pyran-3,4,5-triyl
triacetate (17)
was added trifluoroacetic acid (6.0 mL). The mixture was stirred at r.t.
until the white precipitate appeared. The solution was diluted with
EtOAc (30.0 mL), and the organic layer was washed with saturated so-
dium bicarbonate solution (30.0 mL) and brine (15.0 mL) in order, and
Niclosamide (0.47 g, 1.45 mmoL), potassium carbonate (0.35 g, 2.56
mmoL), and tetrabutylammonium bromide (50 mg, 0.15 mmoL) were
taken up in anhydrous DMF (5.0 mL) in presence of 4 Å molecular sieves,
and a solution of 16 (0.3 g, 0.73 mmoL) in DCM (1.0 mL) was added
dried over anhydrous Na
2 4
SO , and finally concentrated under reduced
pressure to yield 0.95 g of 13 as a white solid with high purity.
1
◦
Yield, 81.0%. H NMR (500 MHz, CD
3
OD) δ 8.41 (d, 1H, J = 9.0 Hz),
dropwise at 0 C. Subsequently, the mixture was rise up to r.t. and stirred
8
2
2
1
.34 (d, 1H, J = 2.5 Hz), 8.17 (dd, 1H, J = 9.0, 2.5 Hz), 7.86 (d, 1H, J =
.5 Hz), 7.37 (dd, 1H, J = 9.0, 2.5 Hz), 6.92 (d, 1H, J = 9.0 Hz), 4.33 (s,
overnight before the reaction mixture was quenched with diluted hy-
drochloric acid (1.0 M), and extracted with EtOAc (35.0 mL). The
organic layer was concentrated under diminished pressure to produce
the crude residue, which was purified by silica gel-based chromatog-
raphy with a mixture of EtOAc and petroleum ether (V/V = 1/4) to
1
3
H). C NMR (125 MHz, CD OD) δ 170.2, 169.7, 159.2, 145.2, 141.8,
3
34.7, 129.4, 126.0, 125.7, 125.2, 124.0, 123.9, 120.0, 118.6, 44.9.
+
+
TOF-MS, m/z: [M + H ], calcd for C15
H
12Cl
2
3
N O
5
, 384.0149, Found,
+
+
3
4
84.0133; [M + Na ], Calcd. for C15
H11Cl
2
N
3
O
5
Na , 405.9968, Found,
afford 0.22 g of 17 as a white solid.
1
05.9951.
Yield, 45.0%. H NMR (500 MHz, CDCl
3
) δ 9.70 (s, 1H), 8.78 (d, 1H,
J = 9.0 Hz), 8.37 (d, 1H, J = 2.5 Hz), 8.20 (dd, 1H, J = 9.0, 2.5 Hz), 8.09
(d, 1H, J = 2.5 Hz), 7.50 (dd, 1H, J = 9.0, 2.5 Hz), 7.13 (d, 1H, J = 9.0
Hz), 5.39–5.26 (m, 2H), 5.22 (d, 1H, J = 7.5 Hz), 5.14 (dd, 1H, J = 10.0,
9.0 Hz), 4.25 (dd, 1H, J = 12.5, 5.0 Hz), 4.12 (dd, 1H, J = 12.5, 2.5 Hz),
3.85 (ddd, 1H, J = 10.0, 5.0, 2.5 Hz), 2.04 (s, 3H), 2.00 (s, 6H), 1.90 (s,
4
.1.13. 2-(4-chloro-2-((2-chloro-4-nitrophenyl)carbamoyl)phenoxy)-2-
oxoethan-1-aminium bromide (14)
.3 g of 13 (0.78 mmoL) was dissolved in 33% hydrobromic acid
0
contained in AcOH (3.0 mL) at 0 ℃, and the mixture was stirred at r. t.
for 0.5 h. The solvent was directly concentrated under diminished
3H). 13C NMR (125 MHz, CDCl
) δ 170.4, 170.2, 169.4, 169.1, 162.2,
3
pressure to yield 0.33 g of 14 as a white solid.
153.0, 143.4, 140.8, 133.9, 132.2, 130.4, 125.8, 125.0, 123.8, 123.5,
1
Yield, 91.7%. H NMR (500 MHz, CD
3
OD) δ 8.42 (d, 1H, J = 2.5 Hz),
121.1, 118.5, 101.1, 72.8, 72.4, 71.0, 67.9, 61.7, 20.7, 20.6, 20.6, 20.5.
8
.30–8.23 (m, 2H), 7.94 (d, 1H, J = 2.5 Hz), 7.71 (dd, 1H, J = 8.5, 2.5
13
Hz), 7.40 (d, 1H, J = 8.5 Hz), 4.18 (s, 2H). C NMR (125 MHz, CD
3
OD) δ
4.1.17. 5-chloro-N-(2-chloro-4-nitrophenyl)-2-(((2S,3R,4S,5S,6R)-3,4,5-
trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzamide
(18)
1
1
67.4, 165.1, 147.9, 146.3, 141.6, 133.9, 133.5, 130.6, 130.2, 128.5,
26.5, 126.3, 126.1, 123.8, 41.4.
To a solution of 17 (0.22 g, 0.33 mmoL) dissolved in dry methanol
(3.0 mL) was added a catalytic amount of sodium methoxide (2.0 mg).
4
.1.14. 4-chloro-2-((2-chloro-4-nitrophenyl)carbamoyl)phenyl sulfamate
+
(
15)
To vigorously stirred solution of chlorosulfonyl isocyanate (1.2 mL,
3.9 mmoL) was added dropwise formic acid (0.53 mL, 13.9 mmoL) at
The mixture was stirred at r. t. for 0.5 h, and excessive Dowex 50 (H )
resins were added to neutralize the resulting solution and shaken for
another 10 mins. The solution was filtered and concentrated under
1
0
◦
C. Five mins later, a white precipitate formed. The precipitate in the
diminished pressure to afford 0.16 g of 18 as a white solid.
1
above reaction was re-dissolved in DCM (5.0 mL), and niclosamide (4.5
g, 13.9 mmoL) in a mixture of DCM (20.0 mL) and dry pyridine (2.0 mL)
Yield, 99.0%. H NMR (400 MHz, CD
3
OD) δ 8.68 (d, 1H, J = 8.8 Hz),
8.42 (d, 1H, J = 2.4 Hz), 8.26 (dd, J = 8.8, 2.4 Hz, 1H), 8.10 (d, 1H, J =
2.4 Hz), 7.61 (dd, 1H, J = 8.8, 2.4 Hz), 7.50 (d, 1H, J = 9.0 Hz), 5.20 (d,
1H, J = 8.0 Hz), 3.92 (d, 1H, J = 2.4 Hz), 3.78–3.67 (m, 2H), 3.53 (dt,
◦
was added in order, and then the reaction mixture was stirred at 50 C
for 6.0 hrs. The resulting solution was adjusted with diluted hydro-
chloric acid (1.0 M), and extracted with EtOAc (100.0 mL). The organic
layer was concentrated under reduced pressure to afford a yellow solid.
The crude solid was further purified by silica gel-based chromatography
1
3
2H, J = 14.4, 5.6 Hz), 3.42 (t, J = 8.8 Hz, 1H). C NMR (100 MHz,
CD
3
OD) δ 164.2, 155.8, 145.1, 142.1, 134.9, 132.2, 129.6, 126.0, 125.8,
125.3, 124.0, 123.9, 120.0, 104.0, 78.7, 78.1, 74.5, 71.1, 62.4. TOF-MS,
7

X. He et al.
Bioorganic Chemistry 107 (2021) 104574
+
-
9
m/z: [M – H ], calcd for C19
.2. Cytotoxicity assay
The in vitro anti-proliferative activities of carboplatin, niclosamide
H
17Cl
2
N
2
O
, 487.0317, Found, 487.0303.
shown as the ratio of the densitometric level with drug treatment to that
without drug treatment after normalization to β-actin.
4
4
.5. Xenograft model antitumor assay
and its derivatives (2, 4, 5, 8, 10, 14, 15, 18) were assessed with the
standard MTT assay [46]. A549 cells or NCI-H446 cells or Jurkat cells or
HBE cells were seeded into 96-well plates. After the addition of freshly
prepared concentrations of the tested compounds, the incubation period
was extended to 48.0 hrs. Five concentrations of the appropriate drugs
with four replicates at each concentration were conducted, and each
experiment was performed in triplicate. Removal of the supernatant and
addition of 40 µL of MTT solution (5 mg/mL) was employed in each well.
After re-incubation for another 4 hrs, 100 µL of DMSO was used to
dissolve the formazan crystals formed in each well. Using a Multiskan
MK3 microplate reader (BioTek Elx800, USA), The measurement of the
percentage of cell viability was performed by the absorbance at a
wavelength of 490 nm. Calculation of IC50 values of the tested com-
pounds was carried out by nonlinear regression analysis with GraphPad
Prism 5.0.
Five- to six-week-old male BALB/c mice (Beijing Charles River Lab-
6
oratories (CRL), China) were inoculated subcutaneously with 2 × 10
human small cell lung cancer NCI-H69 in the flank. When the grafted
3
tumor volume reached the average volume of 100 mm , mice were
randomly divided into five groups (n = 5). The treatment regimen was
shown in Table 2. Compounds 1 and 2 dissolved in 0.5% hydroxypropyl
methyl cellulose (HPMC E5, Dow chemical company, U.S.A) were
dosing at 50 mg/kg once a day to the end. As a positive control group,
carboplatin (Qilu Pharmaceutical, China) at 6 mg/kg was administered
i.g. once a week to the end. On day 29, all the tested animals were
executed with euthanasia, and the tumor was stripped off and weighted.
The in vivo antitumor potency was evaluated in terms of tumor growth
inhibition (TGI), which was defined as the following formula: TGI (%) =
[
(tumor weigh of control group-tumor weigh of treated group)/tumor
weight of control group]*100%. Results were statistically evaluated by
Student’s t-test. This in vivo study was approved by the Institutional
Animal Care and Use Committee (IACUC) of the Guangzhou University
of Chinese Medicine (Guangdong, China) and was performed according
to the institutional guidelines.
4
.3. Water solubility assay
The determination of aqueous solubility was carried out according to
the modified HPLC method [48]. HPLC analysis run on Agilent Tech-
nologies 1260 infinity II with the following conditions: isocratic elution
was adopted, and a mixture of CH
3
OH/H
2
O (V/V = 15/85) containing
Declaration of Competing Interest
0
.1% TFA or 0.1% triethylamine selected for an eluent depending on the
tested compounds was used; Flow rate was set as 1.0 mg/mL; Column
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
◦
temperature was 35 C; Detection wavelength was chosen as 330 nM.
Agilent Eclipse Plus column (250 × 4.6 mm, 5 µm) was adopted as an
analytic column. The calibration curves of 8, 10, 14, 15, 2, 1 and 18
were established as our previous method [48]. Finally, enough amounts
of 8, 10, 14, 15, 18, 2 or 1 was dissolved in 0.2 mL of 0.1 M phosphate
Acknowledgements
◦
buffer (pH 7.4), and the solution was gently shaken at 25 C for 24 hrs.
The work is supported in part by financial support from National
Natural Science Foundation of China (No.21772028), Chinese National
Major Project for New Drug Innovation (No. 2019ZX09201-002-006).
After filtered with 450 nM filter membrane, 100 µL of the saturated filter
was diluted to 1.5 mL solution, 10 µL of which was taken for HPLC
analysis. The value of aqueous solubility was calculated by substituting
the obtained peak into the corresponding calibration curve.
Appendix A. Supplementary data
4
.4. Western blotting assay
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2020.104574.
Immunoblotting assay was in accordance with our previous method
5
[
47]. Specifically speaking, 2 × 10 cells/well were seeded into six-well
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