Discovery of novel N-benzylbenzamide derivatives as tubulin polymerization inhibitors with potent antitumor activities

Article abstract of DOI:10.1016/j.ejmech.2021.113316

A series of novel N-benzylbenzamide derivatives were designed and synthesized as tubulin polymerization inhibitors. Among fifty-one target compounds, compound 20b exhibited significant antiproliferative activities with IC₅₀ values ranging from 12 to 27 nM against several cancer cell lines, and possessed good plasma stability and satisfactory physicochemical properties. Mechanism studies demonstrated that 20b bound to the colchicine binding site and displayed potent anti-vascular activity. Notably, the corresponding disodium phosphate 20b-P exhibited an excellent safety profile with the LD₅₀ value of 599.7 mg/kg (i.v. injection), meanwhile, it significantly inhibited tumor growth and decreased microvessel density in liver cancer cell H22 allograft mouse model without obvious toxicity. Collectively, 20b and 20b-P are novel promising anti-tubulin agents with more druggable properties and deserve to be further investigated for cancer therapy.

Full text of DOI:10.1016/j.ejmech.2021.113316

European Journal of Medicinal Chemistry 216 (2021) 113316
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of novel N-benzylbenzamide derivatives as tubulin
polymerization inhibitors with potent antitumor activities
Huajian Zhu a, Wenlong Li a, Wen Shuai a, Yang Liu a, Limei Yang a, Yuchen Tan a,
,
Tiandong Zheng a, Hong Yao a, Jinyi Xu a **, Zheying Zhu b, Dong-Hua Yang c,
,
*
Zhe-Sheng Chen c, Shengtao Xu a
a State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing, 210009,
PR China
b Division of Molecular Therapeutics & Formulation, School of Pharmacy, The University of Nottingham, University Park Campus, Nottingham NG7 2RD, UK
c College of Pharmacy and Health Sciences, St. John’s University, 8000 Utopia Parkway, Queens, New York, 11439, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel N-benzylbenzamide derivatives were designed and synthesized as tubulin polymeri-
zation inhibitors. Among fifty-one target compounds, compound 20b exhibited significant anti-
proliferative activities with IC50 values ranging from 12 to 27 nM against several cancer cell lines, and
possessed good plasma stability and satisfactory physicochemical properties. Mechanism studies
demonstrated that 20b bound to the colchicine binding site and displayed potent anti-vascular activity.
Notably, the corresponding disodium phosphate 20b-P exhibited an excellent safety profile with the LD50
value of 599.7 mg/kg (i.v. injection), meanwhile, it significantly inhibited tumor growth and decreased
microvessel density in liver cancer cell H22 allograft mouse model without obvious toxicity. Collectively,
20b and 20b-P are novel promising anti-tubulin agents with more druggable properties and deserve to
be further investigated for cancer therapy.
Received 18 November 2020
Received in revised form
16 February 2021
Accepted 17 February 2021
Available online 24 February 2021
Keywords:
N-benzylbenzamide
Anti-vascular
Druggability
© 2021 Elsevier Masson SAS. All rights reserved.
Tubulin inhibitors
Antitumor
1. Introduction
binding site. In recent decades, colchicine binding site inhibitors
(CBSIs) have held great potential as a new generation of tubulin
Microtubules are stiff, cylindrical polymers of
a
,
b
-tubulins, and
inhibitors, which can overcome the limitations of existing tubulin
inhibitors such as structure complexity, poor aqueous solubility,
narrow therapeutic index, wide inter-patient variability and high
toxicity [9e18]. Notably, CBSIs exhibit promising ability to rapidly
disrupt existing tumor blood vessels, leading to the collapse of
blood vessels and tumor cell death [19,20].
Several typical small-molecular inhibitors of tubulin targeting
the colchicine binding site were evaluated in clinical trials as
vascular disrupting agents (VDAs) (Fig. 1A). Combretastatin A-4 (1,
CA-4) is a natural cis-stilbene derivative isolated from the bark of
the African willow tree Combretum caffrum [9], which was identi-
fied as a typical CBSI [21]. CA-4 displayed both potent anti-
proliferative activity against multiple human cancer cell lines [22]
and anti-vascular activity [23]. The phosphate CA-4 (CA-4P) had
been evaluated in clinical trials, however, it was discontinued due
to the lack of a meaningful improvement in progression-free sur-
vival (PFS) and unfavorable partial response data [24]. Verubulin
(3), also known as MPC-6827, was a very potent tubulin polymer-
ization inhibitor and demonstrated low nanomolar (nM) potency
the head-to-tail association of heterodimers forms linear pro-
a-b
tofilaments [1,2]. They play diverse roles in cells, such as protein
transport, maintaining cell structure, chromosome segregation and
mitosis [3e5]. Microtubule targeting agents (MTAs) such as pacli-
taxel or vincristine, have been widely used in clinics for the treat-
ment of various cancers [6,7].
Six binding sites for taxane, laulimalide, vinca alkaloid, pir-
onetin, maytansine and colchicine have been identified in micro-
tubules. Inhibitors targeting taxane and laulimalide binding sites
can stabilize microtubules while those targeting other four binding
sites can destabilize microtubules [6,8]. Currently, all FDA approved
drugs that disrupt microtubule polymerization for cancer treat-
ment bind to either the taxane binding site or the vinca alkaloids
* Corresponding author.
** Corresponding author.
E-mail address: cpuxst@cpu.edu.cn (S. Xu).
https://doi.org/10.1016/j.ejmech.2021.113316
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

H. Zhu, W. Li, W. Shuai et al.
European Journal of Medicinal Chemistry 216 (2021) 113316
Fig. 1. A) Typical CBSIs under clinical trials; B) Reported CBSIs by modifying TMP or replacing TMP moiety. The 4-methylthiophenyl moiety of IMB5046 was predicted to act as ring A
in Zheng’s work, however, in this study, we speculated that 2-morpholine-5-nitrophenyl moiety located in the position of TMP acting as ring A of known CBSIs according to our SARs
and docking study below.
against diverse cancer cell lines [25], and it was found to act as a
VDA by inducing rapid shutdown of tumor blood flow [26]. Some
verubulin derivatives have been reported to exhibit potent anti-
tumor efficacy and improved therapeutic index [27e29]. Verubulin
was also evaluated in clinical trials; however, it was discontinued
due to cardiovascular toxicity revealed in phase I and phase II
studies [30]. Plinabulin (4), developed from phenylahistin as a
metabolite isolated from Aspergillus ustus [31], was another typical
CBSI which attracted much attention [32]. Currently, a combination
of plinabulin and docetaxel is being evaluated in clinical phase III
trials for the treatment of non-small cell lung cancer [33].
3,4,5-Trimethoxyphenyl (TMP) is a common moiety which
serves as ring A of classical CBSIs, such as colchicine, combretastatin
A-4, podophyllotoxin. TMP is crucial for interacting with tubulin
and producing the maximum antiproliferative activities [34].
Though modifications on the TMP moiety of CBSIs are usually re-
ported to significantly reduced anticancer potency [35,36], there
are still many reports in which modifying TMP or replacing TMP
with other moieties led to the improvement of activity. In 2017,
Pang et al. introduced a selenium atom to the TMP moiety of iso-
combretastatin A-4 (isoCA-4), the 3-methylseleno derivative
(structure 5 in Fig. 1B) displayed comparable in vitro anti-
proliferative activity to isoCA-4 while the in vivo antitumor activity
was significantly improved [37]. The quinazoline of compound 6
was thought to be a surrogate of TMP moiety when binding to the
colchicine site, and this was supported by its crystal structure
complexed with tubulin [29]. In 2018, Wang et al. reported a
modification on the TMP moiety of lead VERU-111, and compound 7
with a unique 3-methoxybenzo [4,5]-dioxene ring was found to be
2e8 times more potent than VERU-111 against three cancer cell
lines [38]. Our group also discovered a quinoline-chalcone com-
pound 8, and its antiproliferative activity was improved by 8e10
fold when compared to its parent compound bearing a TMP moiety
[39].
methylthiophenyl moiety occupied the position where TMP of
colchicine was located [40], which structurally differs from the
SARs of classical CBSIs [14]. Thus, the binding mode of IMB5046
with tubulin should be further investigated.
Based on our previous works on discovering and developing
novel anticancer agents targeting the tubulin-microtubule system
[39,42e48], we further considered to design and synthesize novel
N-benzylbenzamide derivatives based on IMB5046 with the wish
to obtain promising anti-tubulin agents with more druggable
properties (Fig. 2). We set for ourselves the goal of designing new
analogs with (a) equal or higher potency compared to IMB5046, (b)
substantially low toxicity as tested in mouse models, and (c)
improved plasma stability.
Herein, we would like to report the synthesis of these N-ben-
zylbenzamide derivatives, their SARs and mechanism studies,
evaluation of preliminary druggability, in vitro and in vivo anti-
tumor and anti-vascular activities as well as the illustration of the
2-morpholinphenyl moiety as a structurally novel ring A of CBSIs.
2. Results and discussion
2.1. Synthesis of target N-benzylbenzamide derivatives
In our first-round modification, we investigated the SARs of ring
B. Seventeen compounds 13a-q including two indolyl benzoates
13a and 13b and fifteen N-benzylbenzamides 13c-q were synthe-
sized. As shown in Scheme 1, 2-fluoro-5-nitrobenzoic acid 11 was
prepared by the nitration of the starting material 2-fluorobenzoic
acid 10, which underwent a nucleophilic attack by morpholine to
give 2-morpholino-5-nitrobenzoic acid 12. The esterification or
amidation of intermediate 12 with different indolylcarbinols or
benzylamines yielded target compounds 13a-q.
In the second-round modification, the SARs of C-2 position on
ring A were investigated. Eight compounds 16a-h, of which
different amino groups were substituted at the C-2 position while
ring B was maintained as an isovanillic ring, were synthesized as
shown in Scheme 2. Intermediate 11 underwent nucleophilic sub-
stitutions by different amines to give 14a-h, which were reacted
with TBS-protected benzylamine 15 followed by deprotection of
TBS to yield target compounds 16a-h.
A
novel nitrobenzoate IMB5046 (9, 2-morpholin-4-yl-5-
nitrobenzoic acid 4-methylsulfanylbenzyl ester), which was re-
ported by Zheng et al. in 2016, was found to display both potent
in vitro and in vivo antitumor activities, and mechanism studies
indicated that it bound to the colchicine binding site [40]. IMB5046
is distinct from any other known CBSIs; however, its structure-
activity relationships (SARs) have not been investigated. Though
IMB5046 had a remarkable antitumor activity in several mouse
xenograft models, the ester bond, which may be unstable in vivo,
and the nitro group, which is often associated with mutagenicity
and genotoxicity [41], might complicate the further development of
IMB5046. In addition, its docking study showed that the 4-
In the third-round modification, the effects of C-4 and C-5 po-
sitions on activity was further examined, thus, twenty-six com-
pounds 20a-p and 34a-j were synthesized, of which ring B was an
isovanillic ring and C-2 was substituted with morpholine or
piperidine. As illustrated in Scheme 3, benzoic acids 19a-d in which
R were hydrogen or fluorine were prepared by the reactions of their
2

H. Zhu, W. Li, W. Shuai et al.
European Journal of Medicinal Chemistry 216 (2021) 113316
Fig. 2. The designing concept for N-benzylbenzamide derivatives.
Scheme 1. Reagents and conditions: (a) con. HNO3, con. H2SO4, 0 ꢀC to rt, 2 h, 71.2%; (b) morpholine, dioxane, rt, 2 h, yield 76.4%; (c) 4-indolylcarbinol or 5-indolylcarbinol, EDCI,
DMAP (cat.), DCM, rt, 2 h, 60.3e67.8%; (d) i) various benzylamines, EDCI, HOBt, DMAP (cat.), DCM, rt, 2 h, 42.0e88.0%; ii) when R1 or R2 ¼ OTBS, TBAF, THF, rt, 15 min, 61.5e81.1%.
Scheme 2. Reagents and conditions: (a) various amines, dioxane, rt, 2 h, 70.3e90.3%; (b) i) EDCI, HOBt, DMAP (cat.), DCM, rt, 2 h; ii) TBAF, THF, rt, 15 min, 44.4e62.9% over 2 steps;
when R was N-Boc piperazine, Boc was removed under condition: CF3COOH, DCM, rt, 2 h, 43.9% over 2 steps.
corresponding ethyl 2-fluorobenzoates 18a-b with morpholine or
piperidine and following hydrolysis of the ethyl ester. Benzoic acids
22a-b and 24a-f were prepared by refluxing their corresponding 2-
fluorobenzoic acids with morpholine or piperidine in dioxane
(Scheme 3). 5-(Dimethylamino)-2-morpholinobenzoic acid 27 was
prepared by reductive amination of 5-amino-2-morpholinobenzoic
acid 26 with paraformaldehyde followed by the hydrolysis of ethyl
ester (Scheme 3). Benzoic acids 32a-c in which R were methoxy or
benzyloxy groups were prepared by a different method as shown in
Scheme 4. Benzoic acid pinacol esters 30a-b were prepared by
Miyaura borylations of their corresponding bromobenzenes 29a-b,
followed by oxidation using hydrogen peroxide gave 31a-b, and the
intermediates 31a-b underwent etherification and saponification
to produce benzoic acids 32a-c. Larger R groups were also intro-
duced to the C-5 position of ring A. As shown in Scheme 5, benzoic
acids 33a-c and 33d-g were prepared by reductive aminations and
amidations of intermediate 26, respectively, and 33h-j were pre-
pared by Suzuki coupling reactions of intermediate 30a. All pre-
pared benzoic acids reacted with TBS-protected benzylamine 15
followed by deprotection of TBS to afford their corresponding target
compounds 20a-p and 34a-j.
Finally, considering the excellent antiproliferative activity and
satisfactory druggability of compound 20b, its disodium phosphate
20b-P was prepared as shown in Scheme 6. Dibenzyl phosphoryl
chloride was prepared according to the literature method [49]. 20b-
PBn was prepared by the reaction of 20b with newly prepared
dibenzylphosphoryl chloride, which was then debenzylated and
neutralized with NaOH to afford the target disodium phosphate
20b-P.
2.2. Antiproliferative activities and SARs
We first investigated the SARs of ring B. A431 epidermoid cancer
cell line was chosen to test the antiproliferative activities since it
was the most sensitive cell line in Zheng’s work [40]. As the indole
moiety has long been considered to be a privileged structure in ring
B of CBSIs [50,51], we first evaluated the activities of 13a and 13b, of
which the indole moiety took the place of 4-methylthiophenyl
moiety in IMB5046. However, they all displayed a significant
decrease of activity (IC50 > 1 mM) (Table 1). The following modifi-
cations led to fifteen N-benzylbenzamide derivatives 13c-q by
replacing the ester bond of IMB5046 with amide linkage.
3

H. Zhu, W. Li, W. Shuai et al.
European Journal of Medicinal Chemistry 216 (2021) 113316
Scheme 3. Reagents and conditions: (a) Con. H2SO4 (Cat.), EtOH, overnight, reflux; (b) i) morpholine or piperidine, DMSO, 120 ꢀC, 6 h; ii) 10% NaOH aqueous, CH3OH, 80 ꢀC, 30 min;
44.9e55.4% over 2 steps; (c) method A: i) EDCI, HOBt, DMAP (cat.), DCM, rt, 2 h; ii) TBAF, THF, rt, 15 min, 40.7e59.0% over 2 steps; method B: i) HATU, Et3N, CH3CN, rt, 2 h; ii) TBAF,
THF, rt, 15 min, 39.5e55.7% over 2 steps; (d) i) morpholine or piperidine, dioxane, reflux, 2 h-overnight, ii) neutralized by 10% HCl aqueous, 73.8e93.6% over 2 steps; (e) Fe, EtOH,
AcOH, reflux, 30 min, 76.1%; (f) i) paraformaldehyde, NaBH(CN)3, AcOH, 30 min, rt; ii) 10% NaOH aqueous, CH3OH, 80 ꢀC, 30 min; 65.4% over 2 steps.
Scheme 4. Reagents and conditions: (a) Con. H2SO4 (Cat.), EtOH, overnight, reflux; (b) Bis(pinacolato)diboron, KOAc, PdCl2(CH3CN)2, dioxane, 80 ꢀC, 3 h, 78.5e86.1%; (c) H2O2,
CH3OH, rt, 30 min, 89.7e93.4%; (d) i) NaH, (CH3)2SO4, 0 ꢀC to rt, 30 min; ii) 10% NaOH aqueous, CH3OH, 80 ꢀC, 30 min; 54.6e63.3% over 2 steps; (e) i) HATU, Et3N, CH3CN, rt, 2 h; ii)
TBAF, THF, rt, 15 min; 39.5e54.5% over 2 steps; (f) i) HATU, Et3N, CH3CN, rt, 2 h ii) TBAF, THF, rt, 15 min; iii) PdeC, H2, CH3OH, rt, 2 h, 16.8% over 3 steps.
Unfortunately, the amide linkage replacement of ester bond led to
about a 22-fold decrease in activity (13c vs. IMB5046). Compound
13d in which R2 was substituted with methoxy, was more potent
than compounds in which R2 was substituted with other groups
such as ethoxy (13e), trifluoromethyl (13f), trifluoromethoxy (13g),
dimethylamino (13h), chlorine (13i) and hydroxy (13j). The intro-
duction of methyl into the linkage (13k) led to a roughly 2-fold drop
of potency when compared with 13d. Encouragingly, the
4

H. Zhu, W. Li, W. Shuai et al.
European Journal of Medicinal Chemistry 216 (2021) 113316
Scheme 5. Reagents and conditions: (a) i) Various benzaldehydes, CH3OH, rt, 30 min; ii) NaBH4, CH3OH, rt, 15 min; iii) 10% NaOH aqueous, CH3OH, 80 ꢀC, 30 min; 49.8e66.9% over 3
steps; (b) i) HATU, Et3N, CH3CN, rt, 2 h; ii) TBAF, THF, rt, 15 min, 42.9e57.9% over 2 steps; (c) HATU, Et3N, CH3CN, rt, 2 h; ii) 10% NaOH aqueous, CH3OH, 80 ꢀC, 30 min; 42.5e64.5%
over 2 steps; (d) i) Various bromobenzenes, K2CO3, Pd(PPh3)4, DMF, 90 ꢀC, 2 h, ii) 10% NaOH aqueous, CH3OH, 80 ꢀC, 30 min, 44.3e57.9% over 2 steps.
Scheme 6. Reagents and conditions: (a) NaH, dibenzylphosphoryl chloride, 0 ꢀC to rt, 2 h, 63.8%; (b) i) PdeC, H2, CH3OH, rt, 30 min; ii) NaOH, CH3OH, rt, 30 min, 89.6% over 2 steps.
Table 1
SARs of ring B.
Compd.
R1
R2
R3
R4
A431 IC50 values (
m
M)a
Compd.
R1
R2
R3
R4
A431 IC50 values (m
M)a
13a
13b
13c
13d
13e
13f
13g
13h
13i
e
e
H
H
H
H
H
H
H
e
e
e
H
H
H
H
H
H
H
e
e
H
H
H
H
H
H
H
>1
>1
13j
13k
13l
13m
13n
13o
13p
13q
IMB5046
H
H
F
OCH3
OH
OCH3
e
OH
H
H
H
H
H
OCH3
e
H
CH3
H
H
H
>1
e
OCH3
OCH3
OCH3
OCH3
OCH3
e
0.241 0.015
0.093 0.007
>1
0.045 0.005
>1
>1
>1
SCH3
OCH3
OC2H5
CF3
OCF3
N(CH3)2
Cl
0.221 0.015
0.127 0.010
0.143 0.012
>1
H
e
>1
0.145 0.015
>1
e
e
e
e
e
e
e
e
0.010 0.005
a
IC50 values are indicated as the mean SD of three independent experiments.
5

H. Zhu, W. Li, W. Shuai et al.
European Journal of Medicinal Chemistry 216 (2021) 113316
introduction of fluorine (13l) and hydroxy (13n) increased the po-
tency and 13n was the most potent compound with an IC50 of
45 nM though it was 4.5-fold less potent than IMB5046
(IC50 ¼ 10 nM). Interestingly, the isovanillic ring, which can be
found in numerous known CBSIs, was the most beneficial moiety as
13n. We further introduced moieties with more steric hindrance to
the C-5 position of ring B. As shown in Table 4, compounds 34a-j all
displayed decreased activity while 34h with a biphenyl skeleton
had moderate activity with an IC50 value of 154 nM, indicating that
there might exist an ample space where the C-5 position is located
in the colchicine binding pocket.
ring
B of the N-benzylbenzamides, indicating that the 4-
methylthiophenyl moiety of IMB5046 might act as ring B of
CBSIs, which is contrary to its predicted binding mode [40].
Consequently, the decision was made to maintain the isovanillic
ring as ring B and focus on efforts to improve potency on other
areas of N-benzylbenzamide derivatives.
Five additional cancer cell lines and one human normal hepa-
tocytes L-O2 cell line were chosen for further evaluation of repre-
sentative compounds. As shown in Table 5, all these selected
compounds displayed potent activities against these five cancer cell
lines in nanomolar ranges, and the SARs were generally consistent
with their performances in A431 cells. All these compounds also
had less potent cytotoxicity against L-O2 cells, which indicated that
these N-benzylbenzamide derivatives might have low toxicity. Even
though 16a was the most active compound with IC50 values ranging
from 11 to 29 nM, the existence of an undesired nitro group in the
structure of 16a prompted us to choose another compound 20b,
which had comparable IC50 values (19e27 nM) to those of 16a, for
further biological evaluation. The comprehensive SARs of the newly
designed N-benzylbenzamide derivatives were summarized as
shown in Fig. 3.
Multidrug resistance (MDR) is one of the main reasons for tumor
chemotherapy failure [52]. As IMB5046 was reported to overcome
multidrug resistance [40], we investigated the antiproliferative
efficacy of compound 20b in the drug-resistant and parental sen-
sitive cells by MTT assays, and the results were shown in Table 6.
The drug-resistant indexes (DRIs) of 20b were comparable to those
of CA-4 and were slightly better than those of IMB5046. Except for
the Adriamycin-resistant cell line Bel-7402/ADR, 20b had DRIs of
5.3e10.8 while positive drugs paclitaxel and vincristine showed
92.5e241.0-fold resistances to the drug-resistant cells, indicating
that 20b had a moderate antiproliferative effect on drug resistant
cancer cells.
We further made our efforts to optimize C-2 position of ring A,
which indicated that substitutions at this position exert great in-
fluences on antiproliferative activity (Table 2). When a piperidine
moiety was introduced (16a), the activity was improved by about 4-
fold when compared to the morpholine counterpart 13n. The IC50
of 16a was 12 nM in A431 cells, which was comparable to that of
IMB5046. However, the activity significantly decreased when the
piperidine was changed to pyrrole (16b) or dimethylamine (16d),
while the diethylamine compound 16c still retained potency
(IC50 ¼ 51 nM). The replacement of oxygen in morpholine by ni-
trogen (16e) and sulfone (16g) led to loss of activity while 16f
bearing a thiomorpholine exhibited a slightly decreased activity
(IC50 ¼ 60 nM). Furthermore, compound 16h bearing a secondary
amino group mimicking the structure of morpholine also displayed
loss of activity.
With the above SARs information in hand, we further investi-
gated SARs of the C-4 and C-5 positions of ring A, and found that
substituents at the C-5 position were well-tolerated while those at
the C-4 position were not (Table 3). Unexpectedly, when R1 was
hydrogen, fluorine, bromine, trifluoromethyl and methoxy groups,
the morpholine derivatives were slightly more potent than the
piperidine counterparts (20a vs 20c, 20b vs 20d, 20h vs 20j, 20i vs
20l and 20n vs 20p). Moreover, in the case of morpholine de-
rivatives, halogens (20b, 20g and 20h) substituted at the C-5 po-
sition was favorable for activity, while trifluoromethyl (20i),
dimethylamino (20m) and hydroxy (20o) containing compounds
was slightly less potent. Compound 20n bearing a methoxy group
also exhibited potent activity with an IC50 value of 28 nM. Incor-
poration of nitrogen heterocycles as ring A (20e and 20f) also led to
a significant drop of potency. The C-4 position did not tolerate
modifications as compound 20j bearing a nitro group at C-4 posi-
tion displayed much less potent activity compared to compound
2.3. Physicochemical and drug-like properties of 20b and 20d
Morpholine was a ubiquitous unit in drug discovery, such as
Gefitinib and Linezolid. Aqueous solubility of lipophilic scaffolds is
often improved by the attachment of a morpholine unit, and
morpholine was reported to have improved solubility and reduced
logP when compared to piperidine [53]. Since compound 20b with
a morpholine unit and 20d with a piperidine unit exhibited similar
antiproliferative activities, their physicochemical and drug-like
Table 2
SARs of C-2 position of ring A.
Compd.
R
A431 IC50 values (
m
M)a
Compd.
R
A431 IC50 values (m
M)a
13n
0.045 0.005
16e
>1
16a
16b
0.012 0.002
0.372 0.025
16f
0.060 0.005
16g
>1
16c
16d
0.051 0.025
16h
>1
>1
IMB5046
0.010 0.005
a
IC50 values are indicated as the mean SD of three independent experiments.
6

H. Zhu, W. Li, W. Shuai et al.
European Journal of Medicinal Chemistry 216 (2021) 113316
Table 3
SARs of C-4 and C-5 position of ring A.
Compd.
R1
R2
X
A431 IC50 values (
m
M)a
Compd.
R1
R2
X
A431 IC50 values (m
M)a
13n
16a
16f
20a
20b
20c
20d
20e
20f
NO2
NO2
NO2
H
F
H
H
H
H
H
H
H
H
e
e
H
O
CH2
S
O
O
CH2
CH2
e
0.045 0.005
0.012 0.002
0.060 0.005
0.020 0.002
0.014 0.003
0.030 0.004
0.025 0.004
0.977 0.085
0.118 0.020
0.025 0.003
20h
20i
20j
20k
20l
20m
20n
20o
20p
IMB5046
Br
CF3
H
H
H
NO2
H
H
H
H
H
H
O
O
O
CH2
CH2
O
O
O
0.030 0.003
0.037 0.005
0.224 0.018
0.092 0.010
0.097 0.010
0.105 0.009
0.028 0.004
0.121 0.008
0.057 0.005
0.010 0.005
Br
CF3
N(CH3)2
OCH3
OH
OCH3
e
F
e
e
e
CH2
e
20g
Cl
O
e
a
IC50 values are indicated as the mean SD of three independent experiments.
Table 4
Further SARs of C-5 position of ring A.
Compd.
R
X
n
1
A431 IC50 values (
m
M)a
Compd.
R
X
n
A431 IC50 values (
0.478 0.035
m
M)a
34a
CH2
0.306 0.023
34f
C¼O
1
34b
34c
CH2
CH2
1
1
1.080 0.102
0.315 0.024
34g
34h
CH3
C¼O
1
0
0.220 0.016
0.154 0.013
e
34d
34e
C¼O
C¼O
1
1
>1
34i
34j
e
e
0
0
0.388 0.022
0.215 0.015
0.962 0.085
a
IC50 values are indicated as the mean SD of three independent experiments.
Table 5
Antiproliferative activities of selected compounds against five cancer cell lines and normal human liver cells.
Compd.
IC50 values (
HepG2
m
M)a
K562
HCT-8
MDA-MB-231
H22
L-O2
13n
16a
16f
20a
20b
20c
20d
20g
20h
20i
20n
IMB5046
CA-4
0.061 0.008
0.012 0.002
0.058 0.004
0.048 0.003
0.025 0.003
0.031 0.004
0.026 0.002
0.029 0.004
0.031 0.003
0.062 0.007
0.029 0.004
0.033 0.004
0.010 0.001
0.059 0.005
0.019 0.003
0.049 0.003
0.030 0.005
0.019 0.004
0.030 0.005
0.011 0.001
0.032 0.003
0.024 0.002
0.040 0.005
0.061 0.005
0.010 0.001
0.007 0.002
0.060 0.008
0.011 0.002
0.060 0.004
0.062 0.008
0.020 0.005
0.029 0.003
0.029 0.006
0.025 0.002
0.030 0.003
0.056 0.006
0.027 0.003
0.027 0.003
0.012 0.004
0.057 0.003
0.029 0.004
0.062 0.005
0.055 0.006
0.027 0.004
0.029 0.003
0.086 0.009
0.030 0.004
0.029 0.005
0.057 0.006
0.029 0.003
0.031 0.006
0.015 0.003
0.050 0.008
0.025 0.003
0.055 0.004
0.049 0.007
0.024 0.005
0.029 0.005
0.032 0.006
0.031 0.005
0.024 0.004
0.055 0.007
0.032 0.004
0.025 0.005
0.015 0.003
0.116 0.015
0.120 0.010
0.113 0.011
0.105 0.012
0.112 0.012
0.097 0.009
0.088 0.012
0.095 0.010
0.108 0.012
0.103 0.013
0.052 0.005
0.044 0.008
0.046 0.009
a
IC50 values are indicated as the mean SD of three independent experiments.
7

H. Zhu, W. Li, W. Shuai et al.
European Journal of Medicinal Chemistry 216 (2021) 113316
Fig. 3. The comprehensive SARs of newly designed N-benzylbenzamide derivatives.
Table 6
The IC50 values of compound 20b in different drug-resistant cancer cells.
Compd.
IC50 values (
Bel-7402
m
M)a
Bel-7402/ADR
DRI
HCT-8
HCT-8/Taxol
DRI
K562
K562/VCR
DRI
20b
IMB-5046
CA-4
ADR
Taxol
VCR
0.027 0.003
0.056 0.004
0.011 0.002
0.274 0.086
ND
0.104 0.012
0.268 0.022
0.058 0.004
1.732 0.202
ND
3.9
4.8
5.3
6.3
e
0.020 0.002
0.027 0.002
0.011 0.002
ND
0.028 0.004
ND
0.106 0.009
0.221 0.023
0.087 0.005
ND
2.590 0.122
ND
5.3
8.2
7.9
e
0.019 0.003
0.010 0.001
0.007 0.001
ND
ND
0.021
0.205 0.015
0.297 0.031
0.057 0.005
ND
ND
5.060
10.8
29.7
8.1
e
92.5
e
e
ND
ND
e
241.0
0.004
0.562
bDRI: drug-resistant index ¼ (IC50 of drug resistant cancer cell)/(IC50 of parental cancer cell).
cND: not detected.
a
IC50 values are indicated as the mean SD of three independent experiments.
properties were further assessed with CA-4 as the reference. As
mL) both at pH 2.0 and pH 7.4. Notably, the morpholine derivative
20b had aqueous solubility of 323.2 g/mL at pH 2.0 and 76.6 g/mL
at pH 7.4, while the piperidine derivative 20d showed low solubility
(<1 g/mL) at pH 7.4. Interestingly, the aqueous solubility of 20d
(1108.2 g/mL) under acidic conditions was much greater than 20b
in the same assay, which may be due to its more basic nature and
ability to form a salt at pH 2.0. Compound 20b-P, the disodium
phosphate of 20b, was soluble in water (>1000 mg/mL at pH 2.0 and
shown in Table 7, the reference CA-4 showed low solubility (<1
m
g/
m
m
m
Table 7
m
Physicochemical and drug-like properties of compounds 20b and 20d.
Compd. MWa
clogPb TPSAc Drug-Score Aqueous
logD7.4
f
Solubilitye
mL)
(mg/
7.4). Additionally, the lipophilicity of 20b and 20d were also
assessed by measuring their octanol/water (aqueous buffer pH 7.4)
partition coefficients through the HPLC/UV method [54]. The
logD7.4 value of 20b was lower than that of 20d, which was
consistent with its aqueous solubility (pH 7.4) and predicted clogP.
Moreover, the predicted topological polar surface area (TPSA)
values for 20b and 20d were in a desirable range (a criterion of
<140 Å2). Their drug-score values, which is a value used to judge a
compound’s overall potential to qualify for a drug, were also pre-
dicted by Osiris calculations. The results showed that 20b had a
higher drug-score value than that of 20d and CA-4, indicating that
20b might be more druggable than 20d and CA-4.
pH 2.0
pH 7.4
20b
20d
CA-4
20b-P
360.0 1.87
358.0 3.03
316.0 3.24
71.03
61.80
57.15
e
0.82
0.71
0.48
e
323.2
1108.2 <1
<1
>1000
76.6
1.61
2.97
2.68
e
1 [55]
>1000
e
e
a
MW: molecular weight.
b
c
clogP: calculated logarithm of the octanol-water partition coefficient.
tPSA: topological polar surface area, predicted by Osiris calculations in https://
www.organic-chemistry.org/prog/peo/.
e
A general solubility guideline for human oral absorption is < 10 mg/mL, low;
10e60
m
g/mL, moderate; > 60
mg/mL, high.
f
Experimental logarithm of the n-octanol/water distribution coefficient at pH
7.4.
8

H. Zhu, W. Li, W. Shuai et al.
European Journal of Medicinal Chemistry 216 (2021) 113316
Table 8
Inhibition of tubulin polymerizationa and colchicine binding to tubulinb.
2.4. Tubulin polymerization by binding to the colchicine binding
site
Compd.
Inhibition of tubulin polymerizaion
Inhibition of colchicine
binding
To investigate whether these N-benzylbenzamide derivatives
are tubulin targeting agents, a representative compound 20b was
selected to undergo tubulin polymerization assay in vitro. In this
assay, tubulin monomer was self-polymerized to microtubules,
increasing light scattering at 340 nm. Microtubule-destabilizing
agent (MDA) colchicine is known to inhibit self-polymerization of
tubulin. On the contrary, microtubule-stabilizing agent (MSA)
paclitaxel promotes self-polymerization of tubulin. As shown in
Fig. 4, 20b produced a significant inhibition of tubulin polymeri-
zation with a similar action to that of colchicine, indicating that 20b
acted as an MDA. The IC50 values of 20b in inhibiting tubulin
polymerization were further determined as shown in Table 8. The
results showed that 20b exhibited a comparable activity to
(%) inhibition SD
IC50
(
m
M)
1
mM
5 mM
20b
IMB5046
CA-4
3.03 0.31
2.99 0.23
2.64 0.22
6.18 0.50
56.9 4.4
58.0 0.6
80.2 1.5
NDc
82.9 4.7
85.7 2.2
90.3 3.4
NDc
20b-P
a
All Data are presented as the mean SD from three independent experiments.
b
c
Tubulin, 1
mM; [3H]-colchicine, 5 mM; and inhibitors, 1 or 5 mM.
ND ¼ not determined.
the colchicine site, which was quite close to that in the crystal
structure (RMSD value ¼ 0.72 Å), indicating the feasibility of our
docking method. As shown in Fig. 5A and C, the phenolic hydroxyl
of 20b and CA-4 formed hydrogen bonds with the residue Thr179.
The binding mode of 5-fluorine-2-morpholine phenyl moiety was
slightly different from that of TMP. Morpholine located at the po-
sition of a methoxy group of TMP interacted with residues Ala 316,
Ala 317 and Ala 354, and the fluorine pointed towards the deeper
pocket which was surrounded by residues Tyr 202, Leu 242, Ala
250, and Leu 255. The binding mode of IMB5046 with tubulin was
similar to that of 20b (Fig. 5B and D), which was in accordance with
their SARs.
IMB5046 with an IC50 value of 3.03
mM, which was slightly less
potent than that of CA-4 (IC50 ¼ 2.64
m
M). In addition, 20b was
examined for its potential inhibition of the binding of [3H]-
colchicine to tubulin. The binding potency of 20b to the colchi-
cine binding site was 82.9% at 5 mM while those of IMB5046 and CA-
4 were 85.7% and 90.3%, respectively, at the same concentration
(Table 8), suggesting that 20b bound to the colchicine binding site
almost as well as IMB5046 and CA-4. Besides, the IC50 value of 20b-
phosphate in inhibiting tubulin polymerization was 6.18 0.50 mM.
2.5. Docking profiles of compound 20b with tubulin
2.6. Anti-microtubule effects in A431 cancer cells
In Zheng’s work, the 4-methylthiophenyl moiety of IMB5046
was predicted to act as ring A resembling the TMP of colchicine
[40]. However, our SARs have showed that the isovanillic ring was
the most beneficial moiety when replacing the 4-methylthiophenyl
of IMB5046, indicating that the 4-methylthiophenyl moiety might
act as ring B of CBSIs. In addition, C-5 position on ring A of N-
benzylbenzamide tolerated modifications with even more steri-
cally demanding moieties introduced, indicating that there might
exist an ample space where the C-5 position fits in the colchicine
binding pocket. As reported before [56], a hydrophobic pocket,
Immunofluorescent staining assay was further performed in
A431 cells to evaluate the inhibitory effects of 20b on microtubule
organization at the cytological level. As shown in Fig. 6, the
microtubule in vehicle-treated A431 cells formed an intact network
with fine filaments. However, cells treated with 20 b at various
concentrations (7.5 nM, 15 nM, and 30 nM) for 24 h resulted in
significant disruption of microtubule networks, loss of cellular
structure, and formation of cell membrane rounding. These results
indicated that 20b significantly caused microtubule network
disruption at the cytological level.
named as zone 3, was buried deeper in the b-subunit of tubulin,
which could be occupied by some CBSIs [44,57,58]. Thus, we
speculated that 4-methylthiophenyl of IMB5046 resembles the
binding mode of isovanillic ring of ring B while the 2-morpholine-
5-nitrophenyl moiety located in the position of TMP acting as ring A
of known CBSIs. To further support this hypothesis, docking studies
of compound 20b and IMB5046 with tubulin were performed using
the Glide module in the Schrodinger software. The original ligand
CA-4 in the crystal structure (PDB:5lyj) [59] was first redocked into
2.7. Cell cycle analysis
Most microtubule polymerization inhibitors interrupt the
tubulin-microtubule balance thus causing mitotic arrest at the G2/
M phase, and ultimately leading to apoptosis [60]. Therefore, the
effects of 20b on cell cycle progression of A431 cells were evaluated
by flow cytometry. As illustrated in Fig. 7A and C, treatment of
20 b at concentrations of 7.5, 15, and 30 nM caused a rise of cells in
G2/M population from 7.27% (control) to 13.84%, 16.25%, and
24.95%, respectively, indicating that 20b arrested A431 cell cycle at
the G2/M phase. Effects of 20b on the expression of cell cycle reg-
ulatory proteins were further tested and the results were shown in
Fig. 7B and D. It was found that 20b decreased cdc2, cyclin B1 and p-
histone H3 protein levels in a concentration-dependent manner,
suggesting that the 20b-induced G2/M arrest may be correlated
with the regulation of cdc2, cyclin B1 and p-Histone H3 protein.
2.8. Cell apoptosis assay
Mitotic arrest of tumor cells by tubulin-targeted agents is
generally associated with cellular apoptosis. Thus, the effects of 20b
on A431 cell apoptosis were examined by fluorescent microscopy in
Hoechst 33342 assays. As shown in Fig. 8A and C, A431 cells treated
with 20b (7.5 nM, 15 nM, and 30 nM) for 48 h displayed significant
Fig. 4. Effects of 20b on tubulin polymerization in vitro. After incubation of purified
tubulin protein with tested compounds, polymerizations were followed by an increase
in absorbance at 340 nm over a 60 min period at 37 ꢀC. The experiments were per-
formed three times.
9

H. Zhu, W. Li, W. Shuai et al.
European Journal of Medicinal Chemistry 216 (2021) 113316
Fig. 5. Proposed binding models for 20b and IMB5046 with tubulin (PDB code: 5lyj). (A) CA-4 (shown in yellow) and 20b (magenta); (B) 20b (shown in magenta) and IMB5046
(green); (C) The 2D interactions between 20b and tubulin; (D) The 2D interactions between IMB5046 and tubulin.
changes in cell morphology, such as nucleus fragmentation and
chromatin condensation. The results of flow cytometry analysis in
Annexin-V/PI assays also indicated that 20b induced A431 cell
apoptosis in a dose-dependent manner (Fig. 8B and D). A431 cells
treated with 20 b at concentrations of 7.5, 15, and 30 nM showed an
accumulation of apoptotic cells (Annexin-Vþ/PIꢁ and Annexin-Vþ/
PIþ) from 2.84% (control) to 20.73%, 44.78%, and 66.14%,
respectively.
treatments of 20b. Taken together, 20b-induced cell apoptosis may
be correlated with upregulation of pro-apoptotic proteins, down-
regulation of anti-apoptotic proteins, caspase activation, cleaved
PARP and p53 activation.
2.9. In vitro evaluation of mitochondrial membrane potential and
reactive oxygen species (ROS) generation
As compound 20b could effectively induced A431 cell apoptosis,
we further evaluated its effects on the expression of apoptosis-
related proteins by Western blot analysis. The Bcl-2 family has
been considered as important proteins that control the mitochon-
drial pathway of apoptosis [61]. As shown in Fig. 9A and B, 20b
upregulated the expression of the pro-apoptotic protein Bax and
downregulated the expression of the anti-apoptotic protein Bcl-2 in
a dose-dependent manner. The caspase families are considered to
be markers for cells undergoing apoptosis and can facilitate cellular
disassembly. Caspase-3 is one of the most important “executioner”
caspases, which cleaves many important cellular substrates [62]. As
shown in Fig. 9A and C, 20b produced a concentration-dependent
activation of caspase-3. Furthermore, a significant increase of
caspase-9 was also observed, which interacted with caspase-3 and
activated apoptosis. The downstream substrate poly (ADP-ribose)
polymerase (PARP) of caspase-3 was also observed to be cleaved
after incubations with 20b. Moreover, p53, which can act in the
cytosol and mitochondria to promote apoptosis through
transcription-independent mechanisms [63], was activated after
Mitochondrial membrane potential (MMP) reduction is
considered an early event of apoptosis. The lipophilic cationic JC-1
staining method was used to determine the effect of compound 20b
on MMP of A431 cells. As presented in Fig. 10A and C, with the
concentration of 20b increasing from 0 to 30 nM, the red fluores-
cence intensity decreased from 93.83% to 33.88%, and the green
fluorescence intensity increased from 5.98% to 65.96%, indicating
that 20b decreased the MMP of A431 cells during the apoptosis
process.
Mitochondria is an important source of ROS in cells [64]. To
further explore the mechanism of 20b for inhibiting tumor cells,
the fluorescent probe 20,70-dichlorofluorescein diacetate (DCF-DA)
was used to evaluate the intracellular ROS levels after incubation
with 20b. As shown in Fig. 10B and D, 20b induced intracellular ROS
generation in a dose-dependent manner. The ratio of DCF positive
cells was increased from 6.17% in cells incubated with DMSO to
21.99% in cells incubated with 30 nM of 20b. Collectively, 20b was
able to increase ROS levels of A431 cells and eventually triggered
apoptotic cell death.
10

H. Zhu, W. Li, W. Shuai et al.
European Journal of Medicinal Chemistry 216 (2021) 113316
subcutaneous injection of H22 cells into the right flank of mice.
Paclitaxel (PTX), IMB5046, CA-4 and its disodium phosphate pro-
drug CA-4P were used as the positive controls. As shown in Fig. 12A
and B, 20b and 20b-P had a significant inhibitory effect on tumor
growth. PTX caused a reduction of tumor weight by 72.7% at a dose
of 6 mg/kg every other day (i.v.) after three weeks as compared to
vehicle, while 20b and 20b-P reduced tumor weights by 66.0% and
71.4% at a dose of 20 mg/kg per day (i.v.), respectively (Fig. 12D).
Particularly, 20b-P (20 mg/kg) displayed more potent antitumor
activity than CA-4P (62.7%) at the same dose. 20b exhibited a
comparable inhibitory rate to IMB5046 at the dose of 20 mg/kg
while that of the disodium phosphate prodrug 20b-P was much
higher. Additionally, PTX caused a significant loss of body weight
(Fig. 12C) while 20b and 20b-P did not obviously affect body weight
even at doses up to 20 mg/kg, indicating the low toxicities of 20b
and 20b-P toward mice. Further H&E staining of some important
origins including heart, liver, spleen, lung and kidney collected
from the 20b-P-treated group (20 mg/kg) also implied low toxicity
of 20b-P (Fig. 13). Taking these results together, 20b and 20b-P
were efficacious and safe in inhibiting the growth of cancer in vivo,
and should be further investigated as potential anticancer agents.
2.13. In vitro and in vivo evaluation of anti-vascular activity
Some microtubule binding drugs possess vascular disrupting
activity due to their disruption ability to microtubule dynamics of
endothelial cells [65]. Thus, wound healing assays was performed
to evaluate the anti-vascular activity of 20b by using human um-
bilical vein endothelial cells (HUVECs). Standardized scratches
(wounds) were made in confluent monolayers of HUVECs and then
incubated with increasing concentrations (7.5, 15 and 30 nM) of
20b. As shown in Fig. 14A, 20b-treatment resulted in significant
inhibition of migration capacity of HUVECs especially at the dose of
30 nM, which indicated that 20b had significant anti-vascular
activity.
The formation of HUVEC tube is the key step in angiogenesis.
Thus, we further evaluated the anti-vascular activity of 20b by
performing a tube formation assay. As shown in Fig. 14B, HUVECs in
the control group form capillary-like tubules with multicentric
junctions, while treatment with 20 b at the concentration of 30 nM
completely inhibited HUVEC cord formation. The IC50 value of 20b
Fig. 6. Effects of 20b on the cellular microtubule network visualized by
immunofluorescence.
2.10. Plasma stability of compound 20b
To assess whether compound 20b, of which amide linkage was
introduced, had improved stability, the in vitro assay using rat
plasma was performed. As shown in Fig. 11, the positive control
Enalapril quickly degraded within 1 h after incubation with rat
plasma at 37 ꢀC, while 20b and IMB5046 were relatively stable in
the same assay. Consistently, 20b had an improved plasma stability
profile compared to IMB5046. After a 3-h incubation, there
remained 91.5% of 20b and only 72.6% of IMB5046. The unexpected
stability of IMB5046, which bears an ester bond, is most likely
attributable to the steric hindrance adjacent to the ester bond,
which might hinder the esterase-catalyzed hydrolysis reaction.
Nevertheless, the remarkable plasma stability of 20b prompted us
to further investigate its safety profile and in vivo antitumor
efficacy.
against HUVECs after 6-h treatment was 0.12 0.03 mM, indicating
that the in vitro potent anti-vascular activity of 20b was not caused
by its cytotoxicity.
CD31 is commonly used as an endothelial cell marker to high-
light tumor blood vessels reflecting the degree of angiogenesis [66].
To assess whether 20b could exert anti-vascular effects in vivo,
immunohistochemical staining against CD31 was performed to
quantify the microvessel density (MVD) of tumors treated with
vehicle and 20b (20 mg/kg). The 20b-treated group exhibited
significantly lower tumor microvessel density (MVD) as compared
with the control group (Fig. 14C), suggesting that 20b exhibited
potent anti-vascular activity in vivo.
2.11. The safety profile of 20b in mice
To understand the safety of 20b, the acute toxicity of its diso-
dium phosphate prodrug 20b-P was assessed in ICR mice. 20b-P
dissolved into saline was intravenously injected at doses of 327.7,
409.6, 512.0, 640, and 800 mg/kg (n ¼ 10 per group) and mice were
observed during two weeks. As shown in Table 10, treatment with
20b-P at a dose of 327.7 mg/kg only caused one death in ten mice,
while treatment with 20b-P at 800 mg/kg killed eight mice. Finally,
the median lethal dose (LD50) value of 20b-P was calculated to be
599.7 mg/kg, implying the low toxicity of 20b-P.
3. Conclusions
With the aim of discovering novel drug candidate with better
druggable profiles as well as illustrating their binding modes with
tubulin, we have performed a comprehensive SARs study of N-
benzylbenzamide derivatives based on the structure of the lead
compound IMB5046. These compounds have a new type of A ring
segment with better drug-like properties, replacing the traditional
3,4,5-trimethoxyphenyl group. Due to the simplicity in the struc-
ture, the target compounds can be obtained through a convenient
and efficient synthetic route from cheap and easily available
2.12. In vivo anti-tumor activity of compound 20b and 20b-P
In order to verify the potential antitumor effect of 20b and 20b-
P in vivo, a liver cancer allograft mouse model was established by
11

H. Zhu, W. Li, W. Shuai et al.
European Journal of Medicinal Chemistry 216 (2021) 113316
Fig. 7. Compound 20b induced G2/M arrest in A431 cancer cells. (A) The percentages of cells at different phases of the cell cycle after treatment with various concentrations of 20b.
(B) Western blotting analysis on the effect of 20b on the G2/M regulatory proteins. (C) Histograms display the percentage of cell cycle distribution. (D) Histograms display the
density ratios of cdc2, cdc25c and p-histone-H3 to GADPH. **p < 0.01, ***p < 0.001 vs. control group.
starting materials. As a result, the representative compound 20b
was eventually found to exhibit potent antiproliferative activity
comparable to IMB5046 with the IC50 values ranging from 12 to
27 nM against six cancer cell lines. Compound 20b displayed a
moderate selectivity for human normal hepatocytes LO2 cells, and
its antiproliferative efficacies against three drug-resistant cancer
cells were also evaluated with the DRIs comparable to those of CA-4
and slightly better than those of IMB5046. Compound 20b, which
has a morpholine unit, and compound 20d, which has a piperidine
unit, were further assessed for their physicochemical and drug-like
properties. Our experiments showed that 20b had satisfactory
physicochemical parameters and its drug-score value was pre-
dicted to be higher than that of 20d or CA-4.
In addition, compound 20b significantly inhibited tubulin
polymerization and disrupted microtubule networks of A431 cells,
and it was further confirmed that 20b binds to the colchicine
binding pocket by colchicine competition inhibition assay and
molecular docking studies through colchicine competition inhibi-
tion assay and molecular docking studies. Notably, our SARs and
docking studies suggested that the binding mode of 20b resembles
that of classical CBSI CA-4 with the 5-fluorine-2-morpholinephenyl
moiety proposed to be a novel ring A of CBSIs. Moreover, 20b
induced A431 cell cycle arrest at the G2/M phase by regulation of
G2/M-related protein expression of cdc2, cyclin B1 and p-Histone
H3 protein. 20b also induced A431 cell apoptosis, which might be
the resulting effects of upregulation of pro-apoptotic protein,
downregulation of anti-apoptotic protein, caspase activation,
cleaved PARP and p53 activation. Additionally, 20b depolarized the
mitochondria membrane potentials and induced ROS generation in
A431 cells.
Furthermore, 20b possessed good plasma stability
(remaining ¼ 91.5% after 3-h incubation) and safety profiles (LD50
value of acute toxicity ¼ 599.7 mg/kg). The antitumor effects of 20b
and its disodium phosphate 20b-P were validated in liver cancer
xenograft mouse model without obvious toxicity. Compounds 20b
and 20b-P reduced tumor weights by 66.0% and 71.4% at a dose of
20 mg/kg per day (iv.), respectively, which were more potent than
the reference CA-4. Finally, the anti-vascular activity of 20b was
confirmed in in vitro HUVEC wound healing and tube formation
assays as well as in vivo microvessel formation in xenograft tumor
tissues. Therefore, 20b and 20b-P deserved further investigations
as novel, safe and efficacious anti-tubulin drug candidates for the
treatment of cancers.
12

H. Zhu, W. Li, W. Shuai et al.
European Journal of Medicinal Chemistry 216 (2021) 113316
Fig. 8. Compound 20b induced apoptosis in A431 cancer cells. (A) Cell morphological alterations and nuclear changes (white arrows) associated with A431 cells after incubation
with 20b; (B) A431 cells stained with Annexin V/PI, followed by flow cytometric analysis; (C) Histograms display the percentage of positive cells; (D) Histograms display the
percentage of cell distribution. ***p < 0.001 vs. control group.
4. Experimental
Low- and high-resolution mass spectra (LRMS and HRMS) were
measured on Finnigan MAT 95 spectrometer (Finnigan, Germany).
Purity of all tested compounds was ꢂ95%, as estimated by HPLC
4.1. Chemistry
(SHIMADZU Labsolutions, UV detection at
l
¼ 254 nm) analysis on
4.1.1. General
the Aglilent C18 column (4.6 ꢃ 150 mm, 5
m
m) eluting at 1 mL/min
All commercially available reagents were used without further
purification. Solvents were dried through routine protocols. Flash
column chromatography was carried out on 200e300 mesh silica
gel (Qingdao Haiyang Chemical, China). Reactions were monitored
by thin-layer chromatography (TLC) on 0.25 mm silicagel plates
(GF254) and visualized under UV light. 1H NMR and 13C NMR
spectra were recorded with a Bruker AV-300 spectrometer (Bruker
Company, Germany) in the indicated solvents (CDCl3 or DMSO‑d6,
TMS as internal standard): the values of the chemical shifts are
of menthol and water (80:20), representative HPLC trace see
Supporting Information.
4.1.2. 2-Morpholino-5-nitrobenzoic acid (12)
To a solution of 2-fluorobenzoic acid (10) (5.0 g, 35.7 mmol) in
10 mL concentrated H2SO4, 10 mL concentrated HNO3 was added
dropwise at 0 ꢀC. After stirring for 2 h at room temperature, the
mixture was slowly poured into ice water. The resulting precipitate
were collected by filtration, which were washed by water, and dried
to give 4.7 g 2-fluoro-5-nitrobenzoic acid (11) as yellow solid, yield
expressed in
d values (ppm) and the coupling constants (J) in Hz.
13

H. Zhu, W. Li, W. Shuai et al.
European Journal of Medicinal Chemistry 216 (2021) 113316
Fig. 9. (A) Western blot analysis of apoptosis-related proteins. (B) Histograms display the density ratios of Bax and Bcl-2 to GADPH. (C) Histograms display the density ratios of
caspase 3, caspase 9, p53 and PARP to GADPH. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control group.
71.2%; To the solution of 11 (500 mg, 2.7 mmol) in 10 mL dioxane,
morpholine (1.2 mL, 13.5 mmol) was added in one potion. After
stirring for 2 h at room temperature, the solvent and remaining
morpholine were removed in vacuo. Then, the residues were dis-
solved into water and neutralized by 10% HCl aqueous. The
resulting precipitate were collected, washed by water, and dried to
give 520 mg 2- morpholine-5-nitrobenzoic acid (12) as yellow
(1H-indol-5-yl)methyl 2-morpholino-5-nitrobenzoate (13b).
Yellow solid, 67.8%. 1H NMR (300 MHz, CDCl3)
d
8.59 (d, J ¼ 2.8 Hz,
1H), 8.40e8.28 (m, 1H), 8.19 (dd, J ¼ 9.2, 2.8 Hz, 1H), 7.77 (d,
J ¼ 1.5 Hz, 1H), 7.41 (d, J ¼ 8.3 Hz, 1H), 7.30 (dd, J ¼ 8.4, 1.6 Hz, 1H),
7.24 (t, J ¼ 2.8 Hz, 1H), 6.91 (d, J ¼ 9.2 Hz, 1H), 6.57 (ddd, J ¼ 3.1, 2.1,
1.0 Hz, 1H), 5.47 (s, 2H), 3.69e3.63 (m, 4H), 3.13e3.07 (m, 4H); 13
C
NMR (75 MHz, CDCl3)
d 166.28, 155.78, 139.64, 135.79, 128.22,
solid, yield 76.4%. 1H NMR (300 MHz, DMSO‑d6)
d
8.40 (d, J ¼ 2.9 Hz,
127.89, 127.74, 126.59, 125.13, 123.38, 122.10, 121.41, 117.10, 111.28,
102.85, 68.60, 66.26, 51.57. HR-MS (ESI) m/z: calcd for C20H20N3O5
[MþH]þ 404.1217 found 404.1212.
1H), 8.20 (dd, J ¼ 9.2, 2.9 Hz, 1H), 7.19 (d, J ¼ 9.2 Hz, 1H), 3.72 (d,
J ¼ 4.8 Hz, 4H), 3.28 (d, J ¼ 4.7 Hz, 4H); 13C NMR (75 MHz, DMSO‑d6)
d
167.39, 155.09, 138.17, 127.42, 127.28, 120.86, 117.75, 65.70, 50.95;
ESI-MS m/z 252.1 [M ꢁ H]- 251.1.
4.1.4. General procedures for the preparation of 13c-q
To a solution of 12 (80 mg, 0.32 mmol) in 10 mL anhydrous DCM,
various benzylamines (0.38 mmol), EDCI (296 mg, 1.6 mmol), HOBt
(52 mg, 0.38 mmol) and catalytic amount of DMAP were added. The
mixtures were stirred for 2 h, and extracted with DCM (3 ꢃ 25 mL).
The combined organic layers were then washed with brine, dried
over anhydrous Na2SO4, and concentrated in vacuo to provide the
crude products, which were purified by column chromatography
with petroleum/ethyl acetate (4:1) to give target compounds. For
benzylamines that containing TBS-protected phenolic hydroxyl, the
products obtained in above procedure were dissolved into 10 mL
THF, and TBAF (1.2 e.q) was added. The mixtures were stirred for
15 min, and extracted with DCM (3 ꢃ 25 mL). The combined organic
layers were then washed with brine, dried over anhydrous Na2SO4,
and concentrated in vacuo to provide the crude products, which
were purified by column chromatography with petroleum/ethyl
acetate (2:1) to give target compounds 13j and 13n.
4.1.3. General procedures for the preparation of 13a and 13b
To a solution of 12 (80 mg, 0.32 mmol) in 10 mL anhydrous DCM,
indolylcarbinol (56 mg, 0.38 mmol), EDCI (296 mg, 1.6 mmol) and
catalytic amount of DMAP were added. The mixtures were stirred
for 2 h, and extracted with DCM (3 ꢃ 25 mL). The combined organic
layers were then washed with brine, dried over anhydrous Na2SO4,
and concentrated in vacuo to provide the crude products, which
were purified by column chromatography with petroleum/ethyl
acetate (4:1) to give target compounds 13a and 13b.
(1H-indol-4-yl)methyl 2-morpholino-5-nitrobenzoate (13a).
Yellow solid, 60.3%. 1H NMR (300 MHz, CDCl3)
d
8.62 (d, J ¼ 2.8 Hz,
1H), 8.36 (s, 1H), 8.20 (dd, J ¼ 9.1, 2.8 Hz, 1H), 7.43 (d, J ¼ 7.6 Hz, 1H),
7.30e7.18 (m, 3H), 6.92 (d, J ¼ 9.2 Hz, 1H), 6.73 (d, J ¼ 2.9 Hz, 1H),
5.70 (s, 2H), 3.61 (t, J ¼ 4.6 Hz, 4H), 3.09 (t, J ¼ 4.7 Hz, 4H); 13C NMR
(75 MHz, CDCl3)
d 166.32, 155.94, 139.75, 136.06, 128.47, 127.88,
127.12, 126.89, 124.94, 121.96, 121.33, 120.97, 117.23, 112.02, 100.92,
N-(4-methylthiobenzyl)-2-morpholino-5-nitrobenzamide
72.80, 66.28, 51.66; HR-MS (ESI) m/z: calcd for
C20H20N3O5
(13c). Yellow solid (78 mg, yield 63.5%). 1H NMR (300 MHz, CDCl3)
d
[MþNa]þ 404.1217 found 404.1211.
8.78 (dd, J ¼ 2.9, 1.0 Hz, 1H), 8.60 (t, J ¼ 5.7 Hz, 1H), 8.22 (dd,
14

H. Zhu, W. Li, W. Shuai et al.
European Journal of Medicinal Chemistry 216 (2021) 113316
Fig. 10. A) Effects of 20b on the MMP of A431 cells; (B) The generation of ROS was measured using DCF-DA in combination with FACScan flow cytometry; (C) Histograms display the
FACScan flow cytometry; (D) Histograms display the intracellular ROS contents in the absence or presence of 20b. **p < 0.01, ***p < 0.001 vs. control group.
Table 10
Acute toxicity of 20b-P in mice.
dose (mg/kg) num. of mice total mortality survival (%) LD50 (mg/kg)a
327.7
409.6
512.0
640.0
800.0
10
10
10
10
10
1
2
3
5
8
90
80
70
50
20
599.7
a
The 95% confidence limits: 530.8e677.5 mg/kg.
d
165.01, 155.49, 143.31, 138.60, 134.53, 128.93, 128.18, 127.35,
127.02, 127.00, 119.43, 66.47, 52.80, 43.64, 15.91; HR-MS (ESI) m/z:
calcd for C19H22N3O4S [MþH]þ 388.1326, found 388.1320.
N-(4-methoxybenzyl)-2-morpholino-5-nitrobenzamide
(13d). Yellow solid (103 mg, yield 88.0%). 1H NMR (300 MHz, CDCl3)
d
8.84 (d, J ¼ 2.7 Hz, 1H), 8.50 (s, 1H), 8.24 (dd, J ¼ 9.0, 2.7 Hz, 1H),
7.31 (d, J ¼ 8.3 Hz, 2H), 7.16 (d, J ¼ 8.9 Hz, 1H), 6.90 (d, J ¼ 8.2 Hz,
2H), 4.58 (d, J ¼ 5.6 Hz, 2H), 3.81 (s, 3H), 3.55 (t, J ¼ 4.5 Hz, 4H), 3.01
(t, J ¼ 4.5 Hz, 4H); 13C NMR (75 MHz, CDCl3)
d 164.82, 159.45,
Fig. 11. Stability of 20b and IMB5046 in rat plasma.
155.50, 143.32, 129.84, 129.78, 128.28, 127.36, 126.93, 119.40, 114.40,
66.41, 55.43, 52.77, 43.57; HR-MS (ESI) m/z: calcd for C19H22N3O5
[MþH]þ 372.1554, found 372.1548.
J ¼ 8.9, 2.0 Hz, 1H), 7.31 (d, J ¼ 8.2 Hz, 2H), 7.24 (d, J ¼ 8.1 Hz, 2H),
7.15 (d, J ¼ 8.9 Hz,1H), 4.60 (d, J ¼ 5.8 Hz, 2H), 3.58 (t, J ¼ 4.6 Hz, 4H),
3.02 (t, J ¼ 4.6 Hz, 4H), 2.48 (s, 3H); 13C NMR (75 MHz, CDCl3)
N-(4-ethoxybenzyl)-2-morpholino-5-nitrobenzamide (13e).
Yellow solid (80 mg, yield 77.9%). 1H NMR (300 MHz, CDCl3)
d 8.85
15

H. Zhu, W. Li, W. Shuai et al.
European Journal of Medicinal Chemistry 216 (2021) 113316
Fig. 12. 20b and 20b-P inhibited liver cancer xenograft growth in vivo. Mice were treated with vehicle, PTX (6 mg/kg every other day), 20b (10 or 20 mg/kg per day), 20b-P (20 mg/
kg per day), IMB5046 (20 mg/kg per day), CA-4 (20 mg/kg per day), and CA-4P (20 mg/kg per day) for three weeks, and then sacrificed for analysis. All the groups were administered
by intravenous injection. The treatments started the day after cell inoculum and the statistics started since the tumor grew out on the 9th day. (A) The images of tumors isolated
from the mice at the end of the experiment; (B) Tumor volume changes of mice during treatment; (C) Body weight changes of mice during treatment; (D) The weight of the excised
tumors of each group. ***P < 0.001 vs. control group.
Fig. 13. HE staining of vital organs of mice including heart, liver, spleen, lung and kidney.
(d, J ¼ 2.8 Hz, 1H), 8.51 (s, 1H), 8.25 (dd, J ¼ 8.9, 2.8 Hz, 1H), 7.30 (d,
J ¼ 8.6 Hz, 2H), 7.16 (d, J ¼ 8.9 Hz, 1H), 6.89 (d, J ¼ 8.6 Hz, 2H), 4.58
(d, J ¼ 5.6 Hz, 2H), 4.03 (q, J ¼ 7.0 Hz, 2H), 3.58e3.48 (m, 4H),
3.06e2.97 (m, 4H), 1.42 (t, J ¼ 7.0 Hz, 3H); 13C NMR (75 MHz, CDCl3)
d 164.69, 158.83, 155.51, 143.39, 129.77, 129.73, 128.41, 127.43,
126.90, 119.40, 114.96, 66.42, 63.61, 52.81, 43.59, 14.89; HR-MS (ESI)
m/z: calcd for C20H24N3O5 [MþH]þ 386.1710, found 386.1712.
2-Morpholino-5-nitro-N-(4-(trifluoromethyl)benzyl)
16

H. Zhu, W. Li, W. Shuai et al.
European Journal of Medicinal Chemistry 216 (2021) 113316
Fig. 14. In vitro and in vivo anti-vascular activity of 20b. (A) Scratches were made with 200
m
L sterile pipette tips, and images were taken at 0 h and 24 h with a phase contrast
microscopy after treatments with 0, 7.5, 15 and 30 nM of 20b; (B) HUVEC capillary-like tubular network formation after treatments with 0, 7.5, 15 and 30 nM of 20b for 6 h; (C)
Immunohistochemical staining of CD31 in tumors treated with vehicle or 20b (20 mg/kg). **P < 0.01 vs. control group.
benzamide (13f). Yellow solid (99 mg, yield 81.1%). 1H NMR
(300 MHz, CDCl3)
8.25 (dd, J ¼ 8.9, 2.8 Hz, 1H), 7.63 (d, J ¼ 8.0 Hz, 2H), 7.51 (d,
J ¼ 8.0 Hz, 2H), 7.20 (d, J ¼ 8.9 Hz, 1H), 4.72 (d, J ¼ 6.0 Hz, 2H),
3.70e3.55 (m, 4H), 3.14e2.99 (m, 4H); 13C NMR (75 MHz, CDCl3)
J ¼ 1.8 Hz, 4H), 7.19 (d, J ¼ 8.9 Hz, 1H), 4.63 (d, J ¼ 5.8 Hz, 2H),
3.65e3.53 (m, 4H), 3.09e2.98 (m, 4H); 13C NMR (75 MHz, CDCl3)
d 165.16, 155.51, 143.33, 136.41, 133.94, 129.74, 129.20, 128.01,
d
8.81 (d, J ¼ 2.8 Hz, 1H), 8.77 (t, J ¼ 6.5 Hz, 1H),
127.36, 127.10, 119.53, 66.52, 52.83, 43.38; HR-MS (ESI) m/z: calcd
for C18H19ClN3O4 [MþH]þ 376.1059 found 376.1058.
d
165.19, 155.56, 143.50, 142.09, 130.31 (q, J ¼ 32.6 Hz), 128.54,
N-(4-hydroxybenzyl)-2-morpholino-5-nitrobenzamide (13j).
Yellow solid (65 mg, yield 81.1% over 2 steps). 1H NMR (300 MHz,
128.07, 127.48, 127.18, 125.99 (q, J ¼ 3.8 Hz), 124.05 (q, J ¼ 270.5 Hz),
119.66, 66.58, 52.92, 43.51; HR-MS (ESI) m/z: calcd for
C
DMSO‑d6)
d
9.35 (s, 1H), 9.01 (t, J ¼ 6.0 Hz, 1H), 8.17 (dd, J ¼ 9.0,
19H19F3N3O4 [MþH]þ 410.1322 found 410.1319.
2.9 Hz, 1H), 8.12 (d, J ¼ 2.8 Hz, 1H), 7.15 (t, J ¼ 8.1 Hz, 3H), 6.73 (d,
J ¼ 8.4 Hz, 2H), 4.32 (d, J ¼ 5.9 Hz, 2H), 3.51 (t, J ¼ 4.5 Hz, 4H), 3.09 (t,
2-Morpholino-5-nitro-N-(4-(trifluoromethoxy)benzyl)ben-
zamide (13g). Yellow solid (79 mg, yield 58.6%). 1H NMR (300 MHz,
J ¼ 4.6 Hz, 4H); 13C NMR (75 MHz, DMSO‑d6)
d 166.37, 156.47,
CDCl3)
d
8.86 (d, J ¼ 2.8 Hz, 1H), 8.67 (s, 1H), 8.27 (dd, J ¼ 8.9, 2.8 Hz,
154.38, 139.00, 129.33, 129.15, 126.89, 126.14, 125.50, 117.56, 115.09,
65.53, 50.48, 42.33; HR-MS (ESI) m/z: calcd for C18H20N3O5 [MþH]þ
358.1397 found 358.1396.
1H), 7.43 (d, J ¼ 8.6 Hz, 2H), 7.23 (d, J ¼ 8.6 Hz, 2H), 7.20 (d,
J ¼ 9.0 Hz, 1H), 4.67 (d, J ¼ 5.9 Hz, 2H), 3.62e3.54 (m, 4H),
3.06e2.98 (m, 4H); 13C NMR (75 MHz, CDCl3)
d
165.04, 155.54,
N-(1-(4-methoxyphenyl)ethyl)-2-morpholino-5-
148.93, 143.46, 136.88, 129.84, 128.17, 127.44, 127.10, 121.60, 120.51
(q, J ¼ 255.8 Hz), 119.59, 66.51, 52.89, 43.25; HR-MS (ESI) m/z: calcd
for C19H19F3N3O5 [MþH]þ 426.1271, found 426.1267.
nitrobenzamide (13k). Yellow solid (99 mg, yield 81.1%). 1H NMR
(300 MHz, CDCl3)
d
8.82 (d, J ¼ 2.8 Hz, 1H), 8.43 (d, J ¼ 7.7 Hz, 1H),
8.24 (dd, J ¼ 8.9, 2.8 Hz, 1H), 7.34 (d, J ¼ 8.7 Hz, 2H), 7.16 (d,
J ¼ 8.9 Hz, 1H), 6.92 (d, J ¼ 8.7 Hz, 2H), 5.36e5.27 (m, 1H), 3.82 (s,
3H), 3.64e3.43 (m, 4H), 3.00 (dd, J ¼ 5.5, 3.9 Hz, 4H), 1.61 (d,
N-(4-(dimethylamino)benzyl)-2-morpholino-5-
nitrobenzamide (13h). Yellow solid (99 mg, yield 81.2%). 1H NMR
(300 MHz, CDCl3)
d
8.86 (d, J ¼ 2.8 Hz, 1H), 8.37 (s, 1H), 8.25 (dd,
J ¼ 6.9 Hz, 3H); 13C NMR (75 MHz, CDCl3)
d 164.15, 159.34, 155.47,
J ¼ 8.9, 2.9 Hz, 1H), 7.24 (d, J ¼ 9.4 Hz, 2H), 7.15 (d, J ¼ 8.9 Hz, 1H),
6.71 (d, J ¼ 8.5 Hz, 2H), 4.55 (d, J ¼ 5.5 Hz, 2H), 3.56 (t, J ¼ 4.6 Hz,
4H), 3.08e2.99 (m, 4H), 2.95 (s, 6H); 13C NMR (75 MHz, CDCl3)
143.29, 134.58, 128.46, 127.84, 127.35, 126.85, 119.39, 114.36, 66.32,
55.46, 52.75, 49.02, 21.47; HR-MS (ESI) m/z: calcd for C20H24N3O5
[MþH]þ 343.1401 found 343.1399.
d
164.66, 155.49, 150.49, 143.37, 129.53, 128.57, 127.42, 126.83,
N-(3-fluoro-4-methoxybenzyl)-2-morpholino-5-
125.20, 119.26, 112.85, 66.44, 52.80, 43.76, 40.69; HR-MS (ESI) m/z:
calcd for C20H25N4O4 [MþH]þ 385.1870, found 385.1873.
N-(4-chlorobenzyl)-2-morpholino-5-nitrobenzamide (13i).
nitrobenzamide (13l). Yellow solid (52 mg, yield 42.0%). 1H NMR
(300 MHz, CDCl3)
d
8.82 (s, 1H), 8.62 (s, 1H), 8.25 (d, J ¼ 8.9 Hz, 1H),
7.46e7.03 (m, 3H), 6.95 (t, J ¼ 8.4 Hz, 1H), 4.57 (d, J ¼ 5.8 Hz, 2H),
Yellow solid (91 mg, yield 82.4%). 1H NMR (300 MHz, CDCl3)
d
8.85
3.90 (s, 3H), 3.60 (s, 4H), 3.03 (s, 4H); 13C NMR (75 MHz, CDCl3)
(d, J ¼ 2.8 Hz, 1H), 8.66 (s, 1H), 8.27 (dd, J ¼ 8.9, 2.8 Hz, 1H), 7.34 (d,
d
165.01,161.69,160.45,155.53,154.18,149.95 (d, J ¼ 20.1 Hz),143.57,
17

H. Zhu, W. Li, W. Shuai et al.
European Journal of Medicinal Chemistry 216 (2021) 113316
130.81 (d, J ¼ 7.1 Hz), 128.25, 127.48, 127.10, 124.30 (d, J ¼ 3.4 Hz),
119.58, 116.33, 116.08, 113.76, 66.56, 56.47, 52.93, 43.28; HR-MS
(ESI) m/z: calcd for C19H21FN3O5 [MþH]þ 390.1460, found 390.1467.
N-(3,4-dimethoxybenzyl)-2-morpholino-5-nitrobenzamide
(13m). Yellow solid (85 mg, yield 66.9%). 1H NMR (300 MHz, CDCl3)
DMSO‑d6)
d
168.51, 151.04, 135.17, 127.59, 126.96, 117.24, 114.34,
51.42, 25.70; ESI-MS m/z 236.1 [M ꢁ H]- 235.1.
2-(Diethylamino)-5-nitrobenzoic acid (14c). Yellow solid,
yield 85.9%. 1H NMR (300 MHz, DMSO‑d6)
d
8.34 (d, J ¼ 2.9 Hz, 1H),
8.17 (dd, J ¼ 9.4, 3.0 Hz, 1H), 7.22 (d, J ¼ 9.4 Hz, 1H), 3.40 (q,
d
8.85 (d, J ¼ 2.8 Hz, 1H), 8.55 (s, 1H), 8.26 (dd, J ¼ 8.9, 2.8 Hz, 1H),
J ¼ 7.0 Hz, 4H), 1.12 (t, J ¼ 7.0 Hz, 6H); 13C NMR (75 MHz, DMSO‑d6)
7.17 (d, J ¼ 8.9 Hz, 1H), 6.93 (dt, J ¼ 4.9, 2.0 Hz, 2H), 6.86 (d,
J ¼ 8.6 Hz, 1H), 4.59 (d, J ¼ 5.7 Hz, 2H), 3.89 (s, 3H), 3.88 (s, 3H),
3.61e3.51 (m, 4H), 3.07e2.97 (m, 4H); 13C NMR (75 MHz, CDCl3)
d 168.40, 153.06, 137.84, 127.31, 127.26, 121.67, 118.01, 46.90, 12.32;
ESI-MS m/z 238.1 [M ꢁ H]- 237.1.
2-(Dimethylamino)-5-nitrobenzoic acid (14d). Yellow solid,
d
164.84, 155.49, 149.44, 148.94, 143.39, 130.35, 128.27, 127.40,
yield 88.2%. 1H NMR (300 MHz, DMSO‑d6)
d
13.27 (s, 1H), 8.35 (d,
126.97, 120.78, 119.43, 111.68, 111.35, 66.44, 56.09, 56.07, 52.83,
43.98; HR-MS (ESI) m/z: calcd for C20H24N3O6 [MþH]þ 402.1660,
found 402.1658.
J ¼ 2.9 Hz, 1H), 8.11 (dd, J ¼ 9.5, 2.9 Hz, 1H), 7.02 (d, J ¼ 9.4 Hz, 1H),
3.03 (s, 6H); 13C NMR (75 MHz, DMSO‑d6)
d 168.26, 154.89, 136.06,
128.05, 127.15, 117.94, 115.35, 42.99; ESI-MS m/z 210.1 [M ꢁ H]-
N-(3-hydroxy-4-methoxybenzyl)-2-morpholino-5-
nitrobenzamide (13n). Yellow solid (75 mg, yield 61.5% over 2
209.1.
2-(4-(Tert-butoxycarbonyl)piperazin-1-yl)-5-nitrobenzoic
acid (14e). Yellow solid, yield 70.3%. 1H NMR (300 MHz, DMSO‑d6)
steps). 1H NMR (300 MHz, CDCl3)
d
8.85 (d, J ¼ 2.8 Hz, 1H), 8.51 (s,
1H), 8.25 (dd, J ¼ 8.9, 2.9 Hz, 1H), 7.17 (d, J ¼ 8.9 Hz, 1H), 6.94 (d,
J ¼ 1.9 Hz, 1H), 6.89e6.81 (m, 2H), 4.55 (d, J ¼ 5.6 Hz, 2H), 3.90 (s,
3H), 3.57 (t, J ¼ 4.7 Hz, 4H), 3.07e3.00 (m, 4H); 13C NMR (75 MHz,
d
8.41 (d, J ¼ 2.9 Hz, 1H), 8.19 (dd, J ¼ 9.2, 2.9 Hz, 1H), 7.17 (d,
J ¼ 9.3 Hz, 1H), 3.48 (t, J ¼ 4.7 Hz, 4H), 3.28 (dd, J ¼ 6.8, 3.6 Hz, 4H),
1.42 (s, 9H); 13C NMR (75 MHz, DMSO‑d6)
167.30, 155.06, 153.83,
d
DMSO‑d6)
d
166.46, 154.37, 146.80, 146.44, 138.95, 131.56, 126.81,
138.06, 127.47, 127.22, 120.62, 117.84, 79.12, 50.22, 27.99; ESI-MS m/
126.17, 125.53, 118.70, 117.51, 115.40, 112.13, 65.57, 55.71, 50.50,
42.39; HR-MS (ESI) m/z: calcd for C19H22N3O6 [MþH]þ 388.1503
found 388.1502.
z 251.1 [M ꢁ H]- 250.1.
5-Nitro-2-thiomorpholinobenzoic acid (14f). Yellow solid,
yield 76.5%. 1H NMR (300 MHz, DMSO‑d6)
d
8.38 (d, J ¼ 3.0 Hz, 1H),
N-(3,4,5-trimethoxybenzyl)-2-morpholino-5-
8.19 (dd, J ¼ 9.3, 3.0 Hz, 1H), 7.21 (d, J ¼ 9.3 Hz, 1H), 3.52 (t,
nitrobenzamide (13o). Yellow solid (63 mg, yield 46.1%). 1H NMR
J ¼ 2.5 Hz, 4H), 2.75 (t, 4H); 13C NMR (75 MHz, DMSO‑d6)
d 167.37,
(300 MHz, CDCl3)
d
8.85 (d, J ¼ 2.8 Hz, 1H), 8.55 (s, 1H), 8.27 (dd,
155.78, 138.26, 127.28, 127.15, 121.60, 118.79, 53.35, 26.31; ESI-MS
J ¼ 8.9, 2.8 Hz, 1H), 7.18 (d, J ¼ 8.9 Hz, 1H), 6.61 (s, 2H), 4.58 (d,
m/z 268.1 [M ꢁ H]- 267.1.
J ¼ 5.8 Hz, 2H), 3.86 (s, 6H), 3.84 (s, 3H), 3.64e3.56 (m, 4H),
2-(1,1-Dioxidothiomorpholino)-5-nitrobenzoic acid (14g).
3.08e2.98 (m, 4H); 13C NMR (75 MHz, CDCl3)
d
164.94, 155.47,
Yellow solid, yield 78.3%. 1H NMR (300 MHz, DMSO‑d6)
d 13.55 (s,
153.74, 143.37, 137.94, 133.58, 128.26, 127.36, 126.99, 119.45, 105.58,
1H), 8.47 (d, J ¼ 2.9 Hz, 1H), 8.25 (dd, J ¼ 9.2, 2.9 Hz, 1H), 7.35 (d,
66.47, 61.03, 56.36, 52.85, 44.45; HR-MS (ESI) m/z: calcd for
J ¼ 9.2 Hz, 1H), 3.76e3.64 (m, 4H), 3.41e3.17 (m, 4H); 13C NMR
C
21H26N3O7 [MþH]þ 432.1765, found 432.1768.
(101 MHz, DMSO) d 167.45, 155.44, 139.80, 127.89, 127.74, 122.26,
2-Morpholino-5-nitro-N-(pyridin-4-ylmethyl)benzamide
120.64, 51.39, 50.35; ESI-MS m/z 300.0 [M ꢁ H]- 209.0.
(13p). Yellow solid (89 mg, yield 82.4%). 1H NMR (300 MHz, CDCl3)
2-((2-Methoxyethyl)amino)-5-nitrobenzoic acid (14h). Yellow
d
8.97e8.76 (m, 2H), 8.60 (d, J ¼ 6.1 Hz, 2H), 8.29 (dd, J ¼ 8.9, 2.8 Hz,
solid, yield 83.2%. 1H NMR (300 MHz, DMSO‑d6)
d 8.87 (s, 1H), 8.63
1H), 7.29 (d, J ¼ 6.1 Hz, 2H), 7.23 (d, J ¼ 8.9 Hz, 1H), 4.68 (d,
(d, J ¼ 2.8 Hz, 1H), 8.16 (dd, J ¼ 9.4, 2.9 Hz, 1H), 6.92 (d, J ¼ 9.5 Hz,
J ¼ 6.0 Hz, 2H), 3.76e3.63 (m, 4H), 3.15e3.03 (m, 4H); 13C NMR
1H), 3.58 (t, J ¼ 5.0 Hz, 2H), 3.51 (t, J ¼ 5.2 Hz, 2H), 3.30 (s, 3H); 13
C
(75 MHz, CDCl3)
d
165.45, 155.56, 150.36, 146.94, 143.43, 127.88,
NMR (75 MHz, DMSO‑d6) d 168.51, 154.63, 134.64, 129.29, 128.51,
127.45, 127.26, 122.79, 119.70, 66.67, 52.93, 42.85; HR-MS (ESI) m/z:
calcd for C17H19N4O4 [MþH]þ 343.1401 found 343.1399.
N-((6-methoxypyridin-3-yl)methyl)-2-morpholino-5-
nitrobenzamide (13q). Yellow solid (79 mg, yield 58.6%). 1H NMR
111.67, 109.14, 69.90, 58.08, 41.99; ESI-MS m/z 240.1 [M ꢁ H]- 239.1.
4-Morpholinonicotinic acid (22a). Yellow solid, yield 88.2%. 1H
NMR (300 MHz, DMSO‑d6)
d 10.00 (s, 1H), 8.60 (s, 1H), 8.40 (d,
J ¼ 7.2 Hz, 1H), 7.48 (d, J ¼ 7.3 Hz, 1H), 3.75 (t, J ¼ 4.5 Hz, 4H), 3.59 (t,
(300 MHz, CDCl3)
d
8.85 (d, J ¼ 2.8 Hz, 1H), 8.58 (s, 1H), 8.27 (dd,
J ¼ 4.5 Hz, 4H); 13C NMR (75 MHz, DMSO‑d6)
d 165.90, 156.79,
143.29, 140.30, 115.45, 112.19, 63.49, 42.89; ESI-MS m/z 208.1
J ¼ 8.9, 2.8 Hz, 1H), 8.17 (d, J ¼ 2.4 Hz, 1H), 7.65 (dd, J ¼ 8.5, 2.5 Hz,
1H), 7.19 (d, J ¼ 8.9 Hz, 1H), 6.79e6.73 (m, 1H), 4.59 (d, J ¼ 5.8 Hz,
2H), 3.94 (s, 3H), 3.65e3.59 (m, 4H), 3.07e3.01 (m, 4H); 13C NMR
[M ꢁ H]- 207.1.
3-Morpholinoisonicotinic acid (22b). Yellow solid, yield 90.3%.
(75 MHz, CDCl3)
d
165.07, 164.04, 155.46, 146.55, 143.29, 139.12,
1H NMR (300 MHz, DMSO‑d6)
d
8.53 (d, J ¼ 3.1 Hz, 1H), 8.32 (d,
128.09, 127.35, 127.03, 126.35, 119.44, 111.35, 66.55, 53.65, 52.78,
40.89; HR-MS (ESI) m/z: calcd for C18H21N4O5 [MþH]þ 373.1506,
found 373.1509.
J ¼ 4.3 Hz, 1H), 7.51 (d, J ¼ 4.4 Hz, 1H), 3.72 (t, 4H), 3.11 (t, J ¼ 4.4 Hz,
4H); 13C NMR (75 MHz, DMSO‑d6)
d 168.12, 144.98, 144.13, 142.79,
132.75, 123.43, 66.62, 52.04; ESI-MS m/z 208.1 [M ꢁ H]- 207.1.
4.1.5. General procedures for the preparation of intermediates 14a-
h and 22a-b
4.1.6. General procedures for the preparation of target compounds
16a-h
The synthesis methods of intermediates 14a-h and 22a-b were
the same as that of 12.
To the solutions of 14a-h (0.32 mmol) in 10 mL anhydrous DCM,
15 (93 mg, 0.64 mmol), EDCI (296 mg, 1.6 mmol), HOBt (52 mg,
0.38 mmol) and catalytic amount of DMAP were added. The mix-
tures were stirred for 2 h, and extracted with DCM (3 ꢃ 25 mL). The
combined organic layers were then washed with brine, dried over
anhydrous Na2SO4, and concentrated in vacuo to provide the crude
products, which were purified by column chromatography with
petroleum/ethyl acetate (4:1) to give products. Then, above ob-
tained products were dissolved into 10 mL THF, and TBAF (1.2 e.q)
was added. The mixtures were stirred for 15 min, and extracted
with DCM (3 ꢃ 25 mL). The combined organic layers were then
washed with brine, dried over anhydrous Na2SO4, and concentrated
5-Nitro-2-(piperidin-1-yl)benzoic acid (14a). Yellow solid,
yield 80.5%. 1H NMR (300 MHz, DMSO‑d6)
d 13.62 (s, 1H), 8.36 (d,
J ¼ 3.0 Hz, 1H), 8.16 (dd, J ¼ 9.2, 3.0 Hz, 1H), 7.19 (d, J ¼ 9.3 Hz, 1H),
3.24 (t, J ¼ 5.2 Hz, 4H), 1.71e1.51 (m, 6H); 13C NMR (75 MHz, DMSO-
d6)
d 167.49, 155.35, 137.66, 127.33, 127.19, 120.78, 117.94, 51.86,
25.14, 23.18; ESI-MS m/z 250.1 [M ꢁ H]- 249.1.
5-Nitro-2-(pyrrolidin-1-yl)benzoic acid (14b). Yellow solid,
yield 76.3%. 1H NMR (300 MHz, DMSO‑d6)
d 13.21 (s, 1H), 8.26 (d,
J ¼ 2.8 Hz, 1H), 8.05 (dd, J ¼ 9.4, 2.8 Hz, 1H), 6.83 (d, J ¼ 9.5 Hz, 1H),
3.29 (t, J ¼ 6.2 Hz, 4H), 1.90 (t, J ¼ 6.1 Hz, 4H); 13C NMR (75 MHz,
18

H. Zhu, W. Li, W. Shuai et al.
European Journal of Medicinal Chemistry 216 (2021) 113316
in vacuo to provide the crude products, which were purified by
column chromatography with petroleum/ethyl acetate (2:1) to give
target compounds 16a-d and 16f-h. For piperazine compound 16e,
the product obtained in above procedure was dissolved into 10 mL
anhydrous DCM, and 2 mL CF3COOH was added. The mixture was
stirred for 2 h, and neutralize with saturated NaHCO3 aqueous. The
mixture was then extracted with DCM (3 ꢃ 25 mL), and the com-
bined organic layers were then washed with brine, dried over
anhydrous Na2SO4, and concentrated in vacuo to provide the crude
products, which were purified by column chromatography with
DCM/MeOH (40:1) to give target compound 16e.
2-(1,1-Dioxidothiomorpholino)-N-(3-hydroxy-4-
methoxybenzyl)-5-nitrobenza mide (16g). Yellow solid, yield
53.4% over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
9.09e9.00 (m,
d
1H), 8.97 (s, 1H), 8.27e8.08 (m, 2H), 7.28 (d, J ¼ 8.8 Hz, 1H), 6.88 (d,
J ¼ 8.2 Hz, 1H), 6.78 (d, J ¼ 2.1 Hz, 1H), 6.73 (dd, J ¼ 8.1, 2.1 Hz, 1H),
4.32 (d, J ¼ 5.9 Hz, 2H), 3.74 (s, 3H), 3.65e3.48 (m, 4H), 3.11 (t,
J ¼ 5.1 Hz, 4H); 13C NMR (75 MHz, DMSO‑d6)
d 166.27, 153.54,
146.79, 146.48, 139.92, 131.58, 127.65, 126.01, 125.41, 119.67, 118.39,
115.10, 112.28, 55.70, 50.61, 49.39, 42.39; HR-MS (ESI) m/z: calcd for
C
19H22N3O7S [MþH]þ 436.1173 found 436.1166.
N-(3-hydroxy-4-methoxybenzyl)-2-((2-methoxyethyl)
N-(3-hydroxy-4-methoxybenzyl)-5-nitro-2-(piperidin-1-yl)
amino)-5-nitrobenzamide (16h). Yellow solid, yield 62.4% over 2
benzamide (16a). Yellow solid, yield 44.4% over 2 steps. 1H NMR
steps. 1H NMR (300 MHz, DMSO‑d6)
d
9.31 (s, 1H), 9.16 (s, 1H), 8.95
(300 MHz, DMSO‑d6)
d
9.00 (t, J ¼ 6.0 Hz, 1H), 8.95 (s, 1H), 8.13 (q,
(s,1H), 8.62 (s,1H), 8.14 (d, J ¼ 9.3 Hz,1H), 6.77 (dd, J ¼ 32.8,16.3 Hz,
J ¼ 3.3 Hz, 2H), 7.16e7.08 (m, 1H), 6.87 (d, J ¼ 8.2 Hz, 1H), 6.80 (d,
J ¼ 2.1 Hz, 1H), 6.73 (dd, J ¼ 8.2, 2.1 Hz, 1H), 4.31 (d, J ¼ 5.9 Hz, 2H),
3.74 (s, 3H), 3.13 (s, 4H), 1.50 (s, 6H); 13C NMR (75 MHz, DMSO‑d6)
4H), 4.32 (s, 2H), 3.75 (s, 3H), 3.56 (s, 2H), 3.45 (s, 2H), 3.30 (s, 3H);
13C NMR (75 MHz, DMSO‑d6)
d 167.30, 154.08, 146.68, 146.44,
134.63, 131.83, 128.12, 125.51, 118.11, 114.86, 113.11, 112.20, 111.07,
d
166.56, 155.06, 146.74, 146.45, 138.05, 131.68, 126.24, 126.17,
70.00, 58.13, 55.71, 54.93, 42.05; HR-MS (ESI) m/z: calcd for
C
125.74, 118.48, 117.56, 115.26, 112.11, 55.71, 51.51, 42.34, 25.08,
23.38; HR-MS (ESI) m/z: calcd for C20H24N3O5 [MþH]þ 386.1710
found 386.1710.
18H22N3O6 [MþH]þ 376.1503 found 376.1496.
4.1.7. General procedures for the preparation of target compounds
20a-p
Target compounds 20a-p were synthesized by two methods.
Method A: the same as the synthesis of 16a-h. Method B: To the
solutions of various benzoic acids (0.32 mmol) in 10 mL anhydrous
CH3CN, 15 (93 mg, 0.64 mmol), HATU (145 mg, 0.38 mmol), and
N-(3-hydroxy-4-methoxybenzyl)-5-nitro-2-(pyrrolidin-1-yl)
benzamide (16b). Yellow solid, yield 50.2% over 2 steps. 1H NMR
(300 MHz, DMSO‑d6)
(m, 4H), 4.46e4.19 (m, 2H), 3.74 (s, 3H), 3.30 (s, 4H), 1.85 (s, 4H); 13
NMR (75 MHz, DMSO‑d6) 167.85, 149.75, 146.75, 146.43, 138.62,
d 8.98 (s, 2H), 8.27e7.77 (m, 2H), 7.17e6.56
C
d
134.36, 131.62, 125.84, 121.48, 118.45, 115.25, 113.36, 112.10, 55.71,
49.85, 42.43, 25.15; HR-MS (ESI) m/z: calcd for C19H22N3O5 [MþH]þ
372.1554 found 372.1551.
Et3N (53 mL, 0.38 mmol) were added. The mixtures were stirred for
2 h, and extracted with DCM (3 ꢃ 25 mL). The combined organic
layers were then washed with brine, dried over anhydrous Na2SO4,
and concentrated in vacuo to provide the crude products, which
were purified by column chromatography with petroleum/ethyl
acetate (4:1) to give products. Then, above obtained products were
dissolved into 10 mL THF, and TBAF was added. The mixtures were
stirred for 15 min, and extracted with DCM (3 ꢃ 25 mL). The
combined organic layers were then washed with brine, dried over
anhydrous Na2SO4, and concentrated in vacuo to provide the crude
products, which were purified by column chromatography with
petroleum/ethyl acetate (2:1) to give target compounds. For com-
pound 20o, the obtained product (56 mg, 0.13 mmol) in above
procedure was dissolved into 10 mL CH3OH, PdeC (5 mg) was
added. The reaction was stirred for 2 h under hydrogen atmo-
sphere, then the mixture was filtered and the filtrate was concen-
trated in vacuo to provide the crude product, which were purified
by column chromatography with petroleum/ethyl acetate (1:1) to
give target compound 20o.
2-(Diethylamino)-N-(3-hydroxy-4-methoxybenzyl)-5-
nitrobenzamide (16c). Yellow solid, yield 55.3% over 2 steps. 1H
NMR (300 MHz, CDCl3)
d
9.09 (t, J ¼ 5.4 Hz, 1H), 8.85 (d, J ¼ 2.9 Hz,
1H), 8.19 (dd, J ¼ 8.9, 2.9 Hz, 1H), 7.16 (d, J ¼ 9.0 Hz, 1H), 6.97e6.88
(m, 1H), 6.83 (d, J ¼ 2.2 Hz, 2H), 5.86 (s, 1H), 4.53 (d, J ¼ 5.4 Hz, 2H),
3.88 (s, 3H), 3.12 (q, J ¼ 7.1 Hz, 4H), 0.98 (t, J ¼ 7.1 Hz, 6H); 13C NMR
(75 MHz, CDCl3)
d 165.07, 154.85, 146.25, 145.99, 142.78, 131.10,
129.04, 127.26, 126.22, 121.87, 119.80, 114.36, 110.92, 56.12, 47.60,
43.77, 11.85; HR-MS (ESI) m/z: calcd for C19H24N3O5 [MþH]þ
374.1710 found 374.1714.
2-(Dimethylamino)-N-(3-hydroxy-4-methoxybenzyl)-5-
nitrobenzamide (16d). Yellow solid, yield 62.9% over 2 steps. 1H
NMR (300 MHz, DMSO‑d6)
2H), 6.89 (t, J ¼ 11.3 Hz, 2H), 6.85e6.45 (m, 2H), 4.29 (d, J ¼ 5.7 Hz,
2H), 3.74 (s, 3H), 2.95 (s, 6H); 13C NMR (75 MHz, DMSO‑d6)
167.61,
d
8.97 (d, J ¼ 7.4 Hz, 2H), 8.31e7.68 (m,
d
153.46, 146.70, 146.41, 135.46, 131.66, 126.02, 125.79, 122.66, 118.38,
115.18, 114.32, 112.11, 55.67, 42.35, 41.89; HR-MS (ESI) m/z: calcd for
C
N-(3-hydroxy-4-methoxybenzyl)-2-morpholinobenzamide
17H20N3O5 [MþH]þ 346.1397 found 346.1393.
(20a). Method A, white solid, yield 54.9% over 2 steps. 1H NMR
N-(3-hydroxy-4-methoxybenzyl)-5-nitro-2-(piperazin-1-yl)
(300 MHz, CDCl3)
d
10.04 (s, 1H), 8.22 (dd, J ¼ 7.8, 1.8 Hz, 1H), 7.44
benzamide (16e). Yellow solid, yield 43.9% over 3 steps. 1H NMR
(td, J ¼ 7.7, 1.8 Hz, 1H), 7.26 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.20 (td, J ¼ 8.0,
1.2 Hz, 1H), 6.97 (d, J ¼ 2.0 Hz,1H), 6.87 (dd, J ¼ 8.2, 2.0 Hz, 1H), 6.82
(d, J ¼ 8.2 Hz, 1H), 6.02 (s, 1H), 4.55 (d, J ¼ 5.3 Hz, 2H), 3.88 (s, 3H),
3.61e3.44 (m, 4H), 2.98e2.85 (m, 4H); 13C NMR (75 MHz, CDCl3)
(300 MHz, DMSO‑d6)
J ¼ 25.3 Hz, 2H), 5.76 (s, 1H), 4.30 (s, 2H), 4.10e3.56 (m, 3H), 3.15 (s,
4H), 2.80 (s, 4H); 13C NMR (75 MHz, DMSO‑d6)
166.49, 154.41,
d 9.04 (s, 2H), 8.15 (s, 2H), 7.15 (s, 1H), 6.82 (d,
d
146.74, 146.43, 138.71, 131.61, 126.54, 126.18, 125.64, 118.48, 117.70,
d 165.99, 150.82, 146.35, 146.13, 132.22, 131.87, 131.60, 127.77,
115.21, 112.12, 55.69, 50.13, 44.20, 42.36; HR-MS (ESI) m/z: calcd for
C
125.29, 120.50, 119.99, 114.70, 110.91, 66.88, 56.11, 53.47, 43.57; HR-
MS (ESI) m/z: calcd for C19H23N2O4 [MþH]þ 343.1652 found
343.1645.
19H23N4O5 [MþH]þ 387.1663 found 387.1664.
N-(3-hydroxy-4-methoxybenzyl)-5-nitro-2-
thiomorpholinobenzamide (16f). Yellow solid, yield 56.0% over 2
steps. 1H NMR (300 MHz, DMSO‑d6)
5-Fluoro-N-(3-hydroxy-4-methoxybenzyl)-2-
morpholinobenzamide (20b). Method B, white solid, yield 46.5%
d
9.02 (t, J ¼ 6.0 Hz, 1H), 8.97 (s,
1H), 8.15 (d, J ¼ 9.3 Hz, 2H), 7.17 (d, J ¼ 8.9 Hz,1H), 6.88 (d, J ¼ 8.1 Hz,
1H), 6.80 (s, 1H), 6.73 (d, J ¼ 8.1 Hz, 1H), 4.31 (d, J ¼ 6.0 Hz, 2H), 3.74
(s, 3H), 3.49e3.38 (m, 4H), 2.60 (d, J ¼ 4.7 Hz, 4H); 13C NMR
over 2 steps. 1H NMR (300 MHz, CDCl3)
d
10.35 (t, J ¼ 5.4 Hz, 1H),
7.96 (dd, J ¼ 9.7, 3.0 Hz, 1H), 7.16 (m, 2H), 6.95 (s, 1H), 6.89e6.79 (m,
2H), 5.96 (s, 1H), 4.53 (d, J ¼ 5.2 Hz, 2H), 3.89 (s, 3H), 3.50 (t,
J ¼ 4.4 Hz, 4H), 2.86 (t, J ¼ 4.5 Hz, 4H); 13C NMR (75 MHz, CDCl3)
(75 MHz, DMSO‑d6) d 166.42, 155.04, 146.76, 146.45, 138.94, 131.63,
127.23, 126.07, 125.54, 118.58, 118.50, 115.24, 112.21, 55.74, 52.96,
42.35, 26.22; HR-MS (ESI) m/z: calcd for C19H22N3O5S [MþH]þ
404.1275 found 404.1277.
d
164.47, 160.16 (d, J ¼ 243.8 Hz), 146.85 (d, J ¼ 3.0 Hz), 146.38,
146.13, 131.37, 130.09 (d, J ¼ 6.9 Hz), 122.93 (d, J ¼ 7.9 Hz), 120.06,
118.83 (d, J ¼ 22.4 Hz), 118.37 (d, J ¼ 24.0 Hz), 114.67, 110.91, 66.86,
19

H. Zhu, W. Li, W. Shuai et al.
European Journal of Medicinal Chemistry 216 (2021) 113316
56.13, 53.70, 43.72; HR-MS (ESI) m/z: calcd for C19H22FN2O4
[MþH]þ 361.1558 found 361.1551.
over 2 steps. 1H NMR (300 MHz, CDCl3)
d 9.39 (s, 1H), 8.43 (d,
J ¼ 2.3 Hz, 1H), 7.68 (dd, J ¼ 8.4, 2.4 Hz, 1H), 7.24 (d, J ¼ 8.4 Hz, 1H),
6.95 (d, J ¼ 1.9 Hz, 1H), 6.93e6.85 (m, 1H), 6.83 (d, J ¼ 8.2 Hz, 1H),
5.83 (s, 1H), 4.55 (d, J ¼ 5.4 Hz, 2H), 3.89 (s, 3H), 3.68e3.36 (m, 4H),
N-(3-hydroxy-4-methoxybenzyl)-2-(piperidin-1-yl)benza-
mide (20c). Method A, white solid, yield 50.5% over 2 steps. 1H NMR
(300 MHz, CDCl3)
d
10.61 (s, 1H), 8.24 (dd, J ¼ 8.1, 1.8 Hz, 1H), 7.40
2.95 (dd, J ¼ 5.5, 3.6 Hz, 4H); 13C NMR (75 MHz, CDCl3)
d 164.92,
(td, J ¼ 7.6, 1.9 Hz, 1H), 7.20 (t, J ¼ 7.7 Hz, 2H), 6.96 (d, J ¼ 2.1 Hz, 1H),
6.86e6.78 (m, 2H), 5.99 (s, 1H), 4.55 (d, J ¼ 5.4 Hz, 2H), 3.86 (s, 3H),
2.81 (d, J ¼ 5.0 Hz, 4H), 1.44 (s, 6H); 13C NMR (75 MHz, CDCl3)
153.51, 146.40, 146.13, 131.25, 129.35 (q, J ¼ 3.7 Hz), 128.90 (q,
J ¼ 3.6 Hz), 128.40, 126.99 (q, J ¼ 33.2 Hz), 123.92 (q, J ¼ 270.5 Hz),
120.31,120.11, 114.61, 110.92, 66.69, 56.15, 53.29, 43.70; HR-MS (ESI)
m/z: calcd for C20H22F3N2O4 [MþH]þ 411.1526 found 411.1519.
N-(3-hydroxy-4-methoxybenzyl)-2-morpholino-4-
d
166.26, 152.69, 146.15, 146.00, 133.43, 131.98, 131.56, 127.66,
124.84, 120.98, 119.84, 114.68, 110.93, 56.16, 54.83, 43.49, 26.24,
23.72; HR-MS (ESI) m/z: calcd for C20H25N2O3 [MþH]þ 341.1860
found 341.1862.
5-Fluoro-N-(3-hydroxy-4-methoxybenzyl)-2-(piperidin-1-yl)
benzamide (20d). Method B, white solid, yield 55.7% over 2 steps.
nitrobenzamide (20j). Method A, white solid, yield 56.5% over 2
steps. 1H NMR (300 MHz, CDCl3)
d
9.34 (s, 1H), 8.31 (d, J ¼ 8.6 Hz,
1H), 8.06 (dd, J ¼ 8.6, 2.1 Hz, 1H), 8.00 (d, J ¼ 2.2 Hz, 1H), 6.96 (d,
J ¼ 1.9 Hz, 1H), 6.91e6.80 (m, 2H), 5.73 (s, 1H), 4.56 (d, J ¼ 5.5 Hz,
2H), 3.91 (s, 3H), 3.54 (t, J ¼ 4.6 Hz, 4H), 3.02e2.91 (m, 4H); 13C NMR
1H NMR (300 MHz, CDCl3)
d
10.79 (s, 1H), 7.97 (dd, J ¼ 9.9, 3.2 Hz,
1H), 7.19 (dd, J ¼ 8.8, 4.9 Hz, 1H), 7.08 (ddd, J ¼ 8.8, 7.2, 3.2 Hz, 1H),
6.95 (d, J ¼ 2.0 Hz, 1H), 6.87 (dd, J ¼ 8.2, 2.0 Hz, 1H), 6.81 (d,
J ¼ 8.2 Hz, 1H), 5.62 (s, 1H), 4.54 (d, J ¼ 5.3 Hz, 2H), 3.88 (s, 3H), 2.79
(d, J ¼ 5.1 Hz, 4H), 1.51e1.42 (m, 6H); 13C NMR (75 MHz, CDCl3)
(75 MHz, CDCl3) d 164.30, 151.56, 149.91, 146.49, 146.18, 133.50,
133.14, 130.92, 120.13, 119.43, 115.21, 114.62, 110.93, 66.55, 56.15,
53.33, 43.77; HR-MS (ESI) m/z: calcd for C19H22N3O6 [MþH]þ
388.1503 found 388.1498.
d
169.92, 159.94 (d, J ¼ 243.4 Hz), 148.69 (d, J ¼ 2.8 Hz), 146.15,
5-Bromo-N-(3-hydroxy-4-methoxybenzyl)-2-(piperidin-1-yl)
benzamide (20k). Method A, white solid, yield 46.3% over 2 steps.
145.98, 139.31, 131.78, 123.20 (d, J ¼ 7.9 Hz), 119.94, 118.57 (d,
J ¼ 22.1 Hz), 117.96 (d, J ¼ 24.0 Hz), 118.12, 117.80, 114.63, 110.90,
56.19, 55.07, 43.61, 26.24, 23.62; HR-MS (ESI) m/z: calcd for
1H NMR (300 MHz, CDCl3)
d
10.34 (s, 1H), 8.36 (d, J ¼ 2.6 Hz, 1H),
7.50 (dd, J ¼ 8.5, 2.6 Hz, 1H), 7.06 (d, J ¼ 8.5 Hz, 1H), 6.94 (d,
J ¼ 2.0 Hz, 1H), 6.86 (dd, J ¼ 8.2, 2.0 Hz, 1H), 6.81 (d, J ¼ 8.2 Hz, 1H),
5.62 (s, 1H), 4.54 (d, J ¼ 5.4 Hz, 2H), 3.88 (s, 3H), 2.80 (d, J ¼ 5.2 Hz,
C
20H24FN2O3 [MþH]þ 359.1765 found 359.1762.
N-(3-hydroxy-4-methoxybenzyl)-4-
morpholinonicotinamide (20e). Method A, white solid, yield
40.7% over 2 steps. 1H NMR (300 MHz, CDCl3)
8.96 (s, 1H), 8.52 (d,
4H), 1.50e1.44 (m, 6H); 13C NMR (75 MHz, CDCl3)
d 164.82, 151.61,
d
146.16, 145.97, 134.75, 134.40, 131.70, 129.58, 122.90, 119.91, 118.32,
114.59, 110.89, 56.19, 54.83, 43.59, 26.15, 23.61; HR-MS (ESI) m/z:
calcd for C20H24BrN2O3 [MþH]þ 419.0965 found 419.0961.
N-(3-hydroxy-4-methoxybenzyl)-2-(piperidin-1-yl)-5-(tri-
fluoromethyl)benzam–ide (20l). Method A, white solid, yield
J ¼ 5.6 Hz,1H), 7.99 (s,1H), 6.94 (d, J ¼ 1.9 Hz,1H), 6.88 (d, J ¼ 5.6 Hz,
1H), 6.85 (d, J ¼ 1.8 Hz, 1H), 6.82 (d, J ¼ 8.1 Hz, 1H), 4.53 (d,
J ¼ 5.7 Hz, 2H), 3.90 (s, 3H), 3.59e3.47 (m, 4H), 3.04e2.93 (m, 4H);
13C NMR (75 MHz, CDCl3)
d 165.69, 156.51, 152.41, 151.94, 146.72,
146.38, 131.05, 122.44, 119.91, 114.78, 112.62, 111.04, 66.36, 56.11,
51.69, 43.52; HR-MS (ESI) m/z: calcd for C18H22N3O4 [MþH]þ
344.1605 found 344.1599.
57.7% over 2 steps. 1H NMR (300 MHz, CDCl3)
d
9.86 (d, J ¼ 6.9 Hz,
1H), 8.47 (d, J ¼ 2.4 Hz,1H), 7.63 (dd, J ¼ 8.5, 2.4 Hz, 1H), 7.23 (s, 1H),
6.94 (d, J ¼ 2.0 Hz, 1H), 6.87 (dd, J ¼ 8.2, 2.0 Hz, 1H), 6.81 (d,
J ¼ 8.2 Hz, 1H), 5.60 (s, 1H), 4.56 (d, J ¼ 5.5 Hz, 2H), 3.88 (s, 3H), 2.88
(t, J ¼ 4.7 Hz, 4H), 1.51 (d, J ¼ 12.4 Hz, 6H); 13C NMR (75 MHz, CDCl3)
N-(3-hydroxy-4-methoxybenzyl)-3-
morpholinoisonicotinamide (20f). Method A, white solid, yield
56.1% over 2 steps. 1H NMR (300 MHz, CDCl3)
d
9.60 (t, J ¼ 5.5 Hz,
d
165.12, 155.37, 146.21, 146.00, 131.56, 129.07 (q, J ¼ 3.9 Hz), 128.65
1H), 8.53 (d, J ¼ 4.3 Hz, 2H), 7.98 (d, J ¼ 5.0 Hz, 1H), 6.97 (s, 1H), 6.85
(s, 2H), 4.54 (d, J ¼ 5.5 Hz, 2H), 3.88 (s, 3H), 3.51 (t, J ¼ 4.4 Hz, 4H),
(q, J ¼ 3.8 Hz), 128.25, 126.29, 120.87, 119.93, 114.57, 110.91, 56.17,
54.73, 43.61, 26.09, 23.60; HR-MS (ESI) m/z: calcd for C21H24F3N2O3
[MþH]þ 409.1734 found 409.1731.
2.99 (t, J ¼ 4.6 Hz, 4H); 13C NMR (75 MHz, CDCl3)
d 164.00, 146.77,
146.68, 146.32, 145.05, 143.09, 134.62, 130.73, 124.40, 119.92, 114.83,
5-(Dimethylamino)-N-(3-hydroxy-4-methoxybenzyl)-2-
morpholinobenzamide (20m). Method B, white solid, yield 46.3%
111.04, 66.62, 56.07, 53.12, 43.65; HR-MS (ESI) m/z: calcd for
C
18H22N3O4 [MþH]þ 344.1605 found 344.1604.
over 2 steps. 1H NMR (300 MHz, CDCl3)
d 10.84 (s, 1H), 7.70 (d,
5-Chloro-N-(3-hydroxy-4-methoxybenzyl)-2-
J ¼ 3.2 Hz, 1H), 7.13 (d, J ¼ 8.8 Hz, 1H), 6.96 (d, J ¼ 2.0 Hz, 1H),
6.89e6.76 (m, 3H), 5.91 (s, 1H), 4.54 (d, J ¼ 5.2 Hz, 2H), 3.88 (s, 3H),
3.47 (d, J ¼ 5.1 Hz, 4H), 2.97 (s, 6H), 2.82 (t, J ¼ 4.6 Hz, 4H); 13C NMR
morpholinobenzamide (20g). Method A, white solid, yield 53.2%
over 2 steps. 1H NMR (300 MHz, CDCl3)
d
9.93 (d, J ¼ 6.6 Hz, 1H),
8.19 (d, J ¼ 2.7 Hz, 1H), 7.39 (dd, J ¼ 8.5, 2.8 Hz, 1H), 7.13 (d,
J ¼ 8.6 Hz, 1H), 6.95 (d, J ¼ 2.0 Hz, 1H), 6.90e6.80 (m, 2H), 5.88 (s,
1H), 4.53 (d, J ¼ 5.3 Hz, 2H), 3.89 (s, 3H), 3.50 (t, J ¼ 4.5 Hz, 4H), 2.87
(75 MHz, CDCl3) d 166.21, 148.43, 146.23, 146.06, 140.19, 131.90,
128.07, 122.42, 119.99, 115.70, 115.08, 114.69, 110.86, 67.13, 56.14,
53.73, 43.65, 40.81; HR-MS (ESI) m/z: calcd for C21H28N3O4 [MþH]þ
386.2074 found 386.2074.
(t, J ¼ 4.6 Hz, 4H); 13C NMR (75 MHz, CDCl3)
d 164.62, 149.28,
146.40, 146.15, 131.99, 131.78, 131.37, 131.15, 129.48, 122.17, 120.06,
N-(3-hydroxy-4-methoxybenzyl)-5-methoxy-2-
114.66, 110.94, 66.79, 56.15, 53.51, 43.70; HR-MS (ESI) m/z: calcd for
C
morpholinobenzamide (20n). Method B, white solid, yield 54.9%
over 2 steps. 1H NMR (300 MHz, CDCl3) d 10.67 (s, 1H), 7.84 (d,
19H22ClN2O4 [MþH]þ 377.1263 found 377.1266.
5-Bromo-N-(3-hydroxy-4-methoxybenzyl)-2-
J ¼ 3.2 Hz, 1H), 7.17 (d, J ¼ 8.8 Hz, 1H), 7.03e6.94 (m, 2H), 6.87 (dd,
J ¼ 8.3, 2.0 Hz, 1H), 6.82 (d, J ¼ 8.2 Hz, 1H), 5.87 (s, 1H), 4.54 (d,
J ¼ 5.2 Hz, 2H), 3.89 (s, 3H), 3.84 (s, 3H), 3.49 (t, J ¼ 4.5 Hz, 4H), 2.85
morpholinobenzamide (20h). Method A, white solid, yield 59.0%
over 2 steps. 1H NMR (300 MHz, CDCl3)
9.86 (s, 1H), 8.33 (d,
d
J ¼ 2.5 Hz, 1H), 7.54 (dd, J ¼ 8.5, 2.5 Hz, 1H), 7.06 (d, J ¼ 8.6 Hz, 1H),
6.94 (s, 1H), 6.90e6.79 (m, 2H), 5.84 (s, 1H), 4.53 (d, J ¼ 5.3 Hz, 2H),
3.89 (s, 3H), 3.50 (t, J ¼ 4.5 Hz, 4H), 2.87 (t, J ¼ 4.5 Hz, 4H); 13C NMR
(t, J ¼ 4.6 Hz, 4H); 13C NMR (75 MHz, CDCl3)
d 165.47, 157.18, 146.29,
146.10, 143.97, 131.68, 128.93, 122.72, 120.02, 118.88, 115.11, 114.68,
110.88, 67.01, 56.14, 55.74, 53.72, 43.69; HR-MS (ESI) m/z: calcd for
(75 MHz, CDCl3)
d
164.56, 149.78, 146.41, 146.15, 134.96, 134.70,
C
20H25N2O5 [MþH]þ 373.1758 found 373.1748.
131.35, 129.68, 122.40, 120.04, 118.78, 114.66, 110.94, 66.75, 56.14,
53.42, 43.69; HR-MS (ESI) m/z: calcd for C19H22BrN2O4 [MþH]þ
421.0757 found 421.0752.
5-Hydroxy-N-(3-hydroxy-4-methoxybenzyl)-2-
morpholinobenzamide (20o) Method B, white solid, yield 16.8%
over 3 steps. 1H NMR (300 MHz, DMSO‑d6)
10.32 (s, 1H), 9.50 (s,
d
N-(3-hydroxy-4-methoxybenzyl)-2-morpholino-5-(tri-
fluoromethyl)benzamide (20i). Method A, white solid, yield 46.6%
1H), 8.96 (s, 1H), 7.37 (s, 1H), 7.20 (s, 1H), 6.88e6.74 (m, 4H), 4.35 (s,
2H), 3.74 (s, 3H), 3.43 (s, 4H), 2.75 (s, 4H); 13C NMR (75 MHz,
20

H. Zhu, W. Li, W. Shuai et al.
European Journal of Medicinal Chemistry 216 (2021) 113316
DMSO‑d6)
d
164.98, 154.38, 146.86, 146.61, 142.56, 131.62, 129.42,
4.1.9. The general procedures for the preparation of intermediates
24a-f
122.92, 118.65, 118.36, 116.46, 115.28, 112.25, 66.18, 55.73, 53.26,
42.38; HR-MS (ESI) m/z: calcd for C19H28N2O5 [MþH]þ 359.1601
found 359.1597.
To the solutions of various 2-fluorobenzoic acids 23a-
d (2.7 mmol) in 20 mL dioxane, morpholine (1.2 mL, 13.5 mmol) or
piperidine (1.1 mL, 13.5 mmol) was added in one potion. The re-
actions were stirred at refluxing temperature, after the reactions
completed, solvents and remaining morpholine or piperidine were
removed in vacuo. Then, the residues were dissolved into water and
neutralized by 10% HCl aqueous. The resulting precipitate were
collected, washed by water, and dried to give intermediates 24a-f.
5-Chloro-2-morpholinobenzoic acid (24a). White solid, yield
N-(3-hydroxy-4-methoxybenzyl)-5-methoxy-2-(piperidin-1-
yl)benzamide (20p). Method B, white solid, yield 39.5% over 2
steps. 1H NMR (300 MHz, CDCl3)
d
11.08 (s, 1H), 7.85 (d, J ¼ 3.2 Hz,
1H), 7.16 (d, J ¼ 8.8 Hz, 1H), 6.98e6.92 (m, 2H), 6.87 (dd, J ¼ 8.2,
2.1 Hz, 1H), 6.81 (d, J ¼ 8.2 Hz, 1H), 5.61 (s, 1H), 4.55 (d, J ¼ 5.3 Hz,
2H), 3.88 (s, 3H), 3.83 (s, 3H), 2.78 (d, J ¼ 5.2 Hz, 4H),1.44 (s, 6H); 13
C
NMR (75 MHz, CDCl3)
d 165.82, 156.82, 146.08, 145.95, 145.86,
132.07, 128.72, 122.95, 119.88, 118.78, 116.32, 114.78, 114.64, 110.88,
84.5%. 1H NMR (300 MHz, DMSO‑d6)
d 15.54 (s, 1H), 7.78 (d,
56.19, 55.70, 55.02, 43.55, 26.35, 23.70; HR-MS (ESI) m/z: calcd for
C
J ¼ 2.7 Hz, 1H), 7.62 (dd, J ¼ 8.8, 2.6 Hz, 1H), 7.50 (d, J ¼ 8.7 Hz, 1H),
21H27N2O4 [MþH]þ 371.1965 found 371.1958.
3.73 (t, J ¼ 4.5 Hz, 4H), 3.01 (t, J ¼ 4.6 Hz, 4H); 13C NMR (75 MHz,
DMSO‑d6)
d 166.62, 149.77, 133.35, 130.48, 129.50, 127.30, 124.33,
66.65, 52.78; ESI-MS m/z 241.1 [M ꢁ H]- 240.1.
4.1.8. The general procedures for the preparation of intermediates
19a-d
5-Bromo-2-morpholinobenzoic acid (24b). White solid, yield
73.8%. 1H NMR (300 MHz, DMSO‑d6)
d 15.47 (s, 1H), 7.96 (d,
To the solutions of 2-fluorobenzoic acid (10) or 2,5-
difluorobenzoic acid (17) (35.7 mmol) in 20 mL EtOH, a catalytic
amount of concentrated H2SO4 was added, and the mixtures were
refluxed overnight. The solvents were then removed and the resi-
dues were extracted with DCM (3 ꢃ 50 mL). The combined organic
layers were then washed with brine, dried over anhydrous Na2SO4,
and concentrated in vacuo to provide 18a or 18b. To the solutions of
intermediates 18a or 18b (29.1 mmol) in 20 mL DMSO, morpholine
(14.3 mL, 145.7 mmol) or piperidine (13.3 mL, 145.7 mmol) were
added. The mixtures were stirred at 120 ꢀC for 6 h, and then diluted
with 50 mL EtOAc, washed with water (20 mL ꢃ 3), saturated brine,
dried over anhydrous Na2SO4, and concentrated in vacuo to afford
crude products. To the solutions of the above crude products in
10 mL CH3OH, 10 mL 10% NaOH aqueous was added and the mix-
tures were stirred at 80 ꢀC for 30 min CH3OH was then removed in
vacuo and acidized with 10% HCl aqueous to pH 3. The precipitates
were collected by filtration, washed with water and dried to afford
19a-d.
J ¼ 2.6 Hz, 1H), 7.76 (dd, J ¼ 8.6, 2.6 Hz, 1H), 7.46 (d, J ¼ 8.7 Hz, 1H),
3.77 (t, J ¼ 4.4 Hz, 4H), 3.05 (t, J ¼ 4.5 Hz, 4H); 13C NMR (75 MHz,
DMSO‑d6)
d 166.54, 150.22, 136.23, 133.47, 127.43, 124.45, 117.34,
66.64, 52.73; ESI-MS m/z 285.0 [M ꢁ H]- 284.0.
2-Morpholino-5-(trifluoromethyl)benzoic acid (24c). White
solid, yield 79.6%. 1H NMR (300 MHz, DMSO‑d6)
d 13.91 (s, 1H), 7.96
(d, J ¼ 2.4 Hz, 1H), 7.80 (dd, J ¼ 8.7, 2.4 Hz, 1H), 7.36 (d, J ¼ 8.6 Hz,
1H), 3.76 (t, J ¼ 4.5 Hz, 4H), 3.13 (t, J ¼ 4.5 Hz, 4H); 13C NMR
(75 MHz, DMSO‑d6)
d
167.89,154.15,129.61 (d, J ¼ 3.8 Hz),128.34 (d,
J ¼ 3.8 Hz), 126.36, 124.33, 121.97 (q, J ¼ 37.0 Hz), 120.26, 66.43,
52.00; ESI-MS m/z 275.0 [M ꢁ H]- 274.0.
2-Morpholino-4-nitrobenzoic acid (24d). Yellow solid, yield
93.4%. 1H NMR (300 MHz, DMSO‑d6)
d 7.81 (s, 1H), 7.79 (s, 2H), 3.69
(t, J ¼ 4.6 Hz, 4H), 3.06 (t, J ¼ 4.6 Hz, 4H); 13C NMR (75 MHz,
DMSO‑d6)
d 168.09, 151.66, 150.04, 132.17, 131.10, 116.60, 114.08,
66.44, 52.02; ESI-MS m/z 252.1 [M ꢁ H]- 251.1.
5-Bromo-2-(piperidin-1-yl)benzoic acid (24e). White solid,
yield 86.8%. 1H NMR (300 MHz, DMSO‑d6)
d 17.90 (s, 1H), 8.08 (d,
2-Morpholinobenzoic acid (19a). White solid, yield 55.4%. 1H
J ¼ 2.6 Hz, 1H), 7.85 (dd, J ¼ 8.6, 2.6 Hz, 1H), 7.69 (d, J ¼ 8.6 Hz, 1H),
3.07 (t, J ¼ 5.2 Hz, 4H),1.73 (q, J ¼ 5.5 Hz, 4H),1.68e1.56 (m, 2H); 13
C
NMR (300 MHz, DMSO‑d6)
d
8.02e7.93 (m, 1H), 7.64 (d, J ¼ 2.9 Hz,
2H), 7.37 (td, J ¼ 5.4, 2.5 Hz, 1H), 3.80 (t, J ¼ 4.6 Hz, 4H), 3.06 (t,
NMR (75 MHz, DMSO‑d6)
d 166.00, 149.89, 136.54, 133.38, 127.62,
J ¼ 4.6 Hz, 4H); 13C NMR (75 MHz, DMSO‑d6)
d 167.29, 150.95,
125.77, 119.95, 53.94, 25.93, 22.63; ESI-MS m/z 283.0 [M ꢁ H]-
134.12, 131.36, 126.58, 125.40, 123.04, 66.79, 53.06; ESI-MS m/z
282.0.
207.1 [M ꢁ H]- 206.1.
2-(Piperidin-1-yl)-5-(trifluoromethyl)benzoic acid (24f).
White solid, yield 83.5%. 1H NMR (300 MHz, DMSO‑d6)
d 15.90 (s,
5-Fluoro-2-morpholinobenzoic acid (19b). White solid, yield
44.9%. 1H NMR (300 MHz, DMSO‑d6)
d
7.75e7.68 (m, 1H), 7.68e7.59
(m, 1H), 7.52e7.39 (m, 1H), 3.76 (t, J ¼ 4.4 Hz, 4H), 3.03 (t, J ¼ 4.5 Hz,
4H); 13C NMR (75 MHz, DMSO‑d6)
1H), 8.03 (d, J ¼ 2.5 Hz, 1H), 7.84 (dd, J ¼ 8.6, 2.5 Hz, 1H), 7.59 (d,
J ¼ 8.6 Hz, 1H), 3.06 (t, J ¼ 5.1 Hz, 4H), 1.71e1.62 (m, 4H), 1.61e1.53
(m, 2H); 13C NMR (75 MHz, DMSO‑d6)
d 167.09, 154.41, 129.99 (d,
d
166.02, 160.09 (d, J ¼ 243.0 Hz),
146.97 (d, J ¼ 3.0 Hz), 127.82 (d, J ¼ 7.5 Hz), 125.85 (d, J ¼ 8.3 Hz),
120.95 (d, J ¼ 21.8 Hz), 117.14 (d, J ¼ 23.3 Hz), 66.74, 53.17; ESI-MS
m/z 225.1 [M ꢁ H]- 224.1.
J ¼ 3.0 Hz), 127.99 (d, J ¼ 3.8 Hz), 126.15, 125.21, 124.32 (q,
J ¼ 32.5 Hz), 122.47, 53.37, 25.83, 23.16; ESI-MS m/z 273.0 [M ꢁ H]-
272.0.
2-(Piperidin-1-yl)benzoic acid (19c). White solid, yield 49.3%.
1H NMR (300 MHz, DMSO‑d6)
d
8.01 (d, J ¼ 7.8 Hz, 1H), 7.69 (d,
J ¼ 8.0 Hz, 1H), 7.66e7.59 (m, 1H), 7.45e7.36 (m, 1H), 3.01 (t,
4.1.10. Ethyl 5-amino-2-morpholinobenzoate (26)
J ¼ 5.2 Hz, 4H), 1.79e1.68 (m, 4H), 1.65e1.50 (m, 2H); 13C NMR
To the solutions of 12 (39.7 mmol) in 20 mL EtOH, a catalytic
amount of concentrated H2SO4 was added, and the mixtures were
refluxed overnight. The solvents were then removed and the resi-
dues were extracted with DCM (3 ꢃ 50 mL). The combined organic
layers were then washed with brine, dried over anhydrous Na2SO4,
and concentrated in vacuo to provide 25. Ethyl 2-morpholino-5-
nitrobenzoate (25) (5 g, 17.9 mmol) was dissolved into the
mixture solvent of 20 mL EtOH and 20 mL AcOH, Fe powder (6 g,
107.2 mmol) was added, and the mixture was stirred at 65 ꢀC for
30 min. Then, the solvent was removed in vacuo, and the residue
was neutralized by saturated NaHCO3 aqueous. The mixture was
(75 MHz, DMSO‑d6)
d 167.14, 150.97, 134.08, 131.20, 127.76, 125.34,
123.47, 54.15, 26.07, 22.68; ESI-MS m/z 205.1 [M ꢁ H]- 204.1.
5-Fluoro-2-(piperidin-1-yl)benzoic acid (19d). White solid,
yield 53.6%. 1H NMR (300 MHz, DMSO‑d6)
d 18.54 (s, 1H), 7.85 (dd,
J ¼ 8.9, 4.8 Hz, 1H), 7.73 (dd, J ¼ 9.1, 3.2 Hz, 1H), 7.55 (td, J ¼ 8.5,
3.2 Hz, 1H), 3.09 (t, J ¼ 5.4 Hz, 4H), 1.76 (p, J ¼ 5.5 Hz, 4H), 1.68e1.58
(m, 2H); 13C NMR (75 MHz, DMSO‑d6)
d 166.01, 160.73 (d,
J ¼ 243.8 Hz), 146.59, 128.05 (d, J ¼ 7.5 Hz), 126.05 (d, J ¼ 8.3 Hz),
120.92 (d, J ¼ 22.5 Hz), 116.92 (d, J ¼ 23.3 Hz), 54.20, 25.95, 22.48;
ESI-MS m/z 223.1 [M ꢁ H]- 222.1.
21

H. Zhu, W. Li, W. Shuai et al.
European Journal of Medicinal Chemistry 216 (2021) 113316
filtrated, and the filtrates was extracted with EtOAc (3 ꢃ 50 mL), the
combined organic layers were then washed with saturated brine,
dried over anhydrous Na2SO4, and concentrated in vacuo to afford
the crude product, which was purified by column chromatography
with petroleum/ethyl acetate (2:1) to give 3.5 g 26 as yellow solid,
(m, 4H), 1.74 (p, J ¼ 5.6 Hz, 4H), 1.67e1.56 (m, 2H), 1.43 (t, J ¼ 7.1 Hz,
3H), 1.37 (s, 12H); 13C NMR (100 MHz, CDCl3)
d 168.77, 154.82,
138.69, 137.96, 122.95, 117.36, 83.62, 60.80, 53.16, 25.97, 24.85,
24.25, 14.38; ESI-MS m/z 359.2 [MþH]þ 360.2.
yield 76.1%. 1H NMR (300 MHz, DMSO‑d6)
d
6.90 (d, J ¼ 8.7 Hz, 1H),
4.1.13. Ethyl 5-hydroxy-2-morpholinobenzoate (31a)
6.71 (s, 1H), 6.67e6.57 (m, 1H), 5.02 (s, 2H), 4.17 (q, J ¼ 8.7 Hz, 2H),
To a solution of 30a (320 mg, 0.89 mmol) in 20 mL CH3OH, 2 mL
30% H2O2 aqueous was added dropwise. The mixture was stirred at
room temperature for 30 min. Then, the CH3OH was removed in
vacuo. The residue was extracted with EtOAc (3 ꢃ 25 mL), and the
combined organic layers were then washed with saturated brine,
dried over anhydrous Na2SO4, and concentrated in vacuo to afford
the crude product, which was purified by column chromatography
with petroleum/ethyl acetate (5:1) to give 208 mg 31a as white
3.59 (s, 4H), 2.73 (s, 4H), 1.34e1.14 (m, 3H); 13C NMR (75 MHz,
DMSO‑d6)
d 168.39, 145.05, 141.21, 129.00, 121.79, 117.56, 114.83,
67.09, 60.71, 53.77, 14.66; ESI-MS m/z 250.1 [MþH]þ 251.1.
4.1.11. 5-(Dimethylamino)-2-morpholinobenzoic acid (27)
To a solution of 26 (200 mg, 0.8 mmol) in 5 mL AcOH, NaBH3CN
(200 mg, 3.2 mmol) was added. The mixture was stirred at room
temperature for 30 min. Then, the solvent was removed in vacuo
and the residue was neutralized by saturated NaHCO3 aqueous. The
mixture was filtrated, and the filtrates was extracted with EtOAc
(3 ꢃ 25 mL), the combined organic layers were then washed with
saturated brine, dried over anhydrous Na2SO4, and concentrated in
vacuo to afford the crude product. To the solution of the above
crude product in 10 mL CH3OH, 10 mL 10% NaOH aqueous was
added and the mixtures were stirred at 80 ꢀC for 30 min CH3OH was
then removed in vacuo and acidized with 10% HCl aqueous to pH 3.
The precipitates were collected by filtration, washed with water
and dried to afford 154 mg 27 as grey solid, yield 65.4% over 2 steps.
solid, yield 93.4%. 1H NMR (300 MHz, CDCl3)
d
7.19 (d, J ¼ 2.9 Hz,
1H), 7.00 (d, J ¼ 8.7 Hz, 1H), 6.95e6.90 (m, 1H), 4.35 (q, J ¼ 7.1 Hz,
2H), 3.85 (t, 4H), 2.96 (t, 4H), 1.38 (t, J ¼ 7.1 Hz, 3H); 13C NMR
(75 MHz, CDCl3)
d 167.93, 151.77, 145.21, 127.55, 121.30, 119.44,
117.41, 67.31, 61.23, 53.49, 24.79; ESI-MS m/z 251.1 [MþH]þ 252.1.
The synthesis method of intermediate 31b was the same as that
of 31a.
Ethyl 5-hydroxy-2-(piperidin-1-yl)benzoate (31b). white
solid, yield 89.7%. 1H NMR (300 MHz, CDCl3)
d
7.21 (dd, J ¼ 9.1,
2.9 Hz,1H), 7.04e6.97 (m,1H), 6.97e6.90 (m,1H), 4.38 (p, J ¼ 7.0 Hz,
2H), 3.92 (s, 1H), 3.01e2.71 (m, 4H), 1.73 (p, J ¼ 5.6 Hz, 4H), 1.57 (q,
J ¼ 5.6, 5.1 Hz, 2H), 1.42 (t, J ¼ 7.1 Hz, 3H); 13C NMR (75 MHz, CDCl3)
1H NMR (300 MHz, DMSO‑d6)
d
17.78 (s,1H), 7.61 (dd, J ¼ 8.9, 4.5 Hz,
1H), 7.34 (d, J ¼ 1.8 Hz, 0H), 7.04 (d, 1H), 3.83 (t, J ¼ 4.8 Hz, 4H), 3.05
d 168.66, 150.72, 146.81, 126.95, 121.05, 119.32, 117.28, 61.28, 54.70,
(t, J ¼ 4.8 Hz, 4H), 2.99 (s, 2H); 13C NMR (75 MHz, DMSO‑d6)
26.35, 24.16, 14.28; ESI-MS m/z 249.1 [MþH]þ 250.1.
d
167.39, 149.47, 139.07, 125.52, 124.47, 117.61, 113.10, 66.88, 53.38,
40.50; ESI-MS m/z 250.1 [M ꢁ H]- 249.1.
4.1.14. The general procedures for the preparation of intermediates
32a-c
4.1.12. Ethyl 2-morpholino-5-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)benzoate (30a)
To the solutions of 31a-b (0.48 mmol) in anhydrous THF, NaH
(29 mg, 0.72 mmol, 60%) was added and stirred at 0 ꢀC under ni-
trogen atmosphere for 15 min. Then, (CH3)2SO4 (0.45 mL,
To the solutions of 28a (35.0 mmol) in 20 mL EtOH, a catalytic
amount of concentrated H2SO4 was added, and the mixtures were
refluxed overnight. The solvents were then removed and the resi-
dues were extracted with DCM (3 ꢃ 50 mL). The combined organic
layers were then washed with brine, dried over anhydrous Na2SO4,
and concentrated in vacuo to provide 29a. To a solution of inter-
mediate 29a (500 mg, 1.59 mmol) in 20 mL dioxane, PdCl2(CH3CN)2
(110 mg, 0.16 mmol), bis(pinacolato)diboron (613 mg, 2,39 mmol),
and KOAc (470 mg, 4.77 mmol) were added. The mixture was
stirred at 80 ꢀC for 3 h. Then, the mixture was filtrated and the
filtrate was concentrated in vacuo to afford the crude product,
which was purified by column chromatography with petroleum/
ethyl acetate (20:1) to give 495 mg 30a as colorless oil, yield 86.1%.
0.53 mmol) or benzyl bromide (63 mL, 0.53 mmol) were added, and
the mixtures were stirred at 0 ꢀC for another 30 min. The mixtures
were extracted with EtOAc (3 ꢃ 25 mL), and the combined organic
layers were washed with saturated brine, dried over anhydrous
Na2SO4, and concentrated in vacuo to afford the crude products. To
the solutions of the above crude products in 10 mL CH3OH, 10 mL
10% NaOH aqueous was added and the mixtures were stirred at
80 ꢀC for 30 min CH3OH was then removed in vacuo and acidized
with 10% HCl aqueous to pH 3. The precipitates were collected by
filtration, washed with water and dried to afford 32a-c.
Ethyl 5-methoxy-2-morpholinobenzoate (32a). White solid,
yield 57.5% over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
d 7.68 (d,
1H NMR (300 MHz, CDCl3)
d
8.14 (d, J ¼ 1.9 Hz, 1H), 7.89e7.81 (m,
J ¼ 8.8 Hz, 1H), 7.47 (d, J ¼ 3.1 Hz, 1H), 7.20 (dd, J ¼ 8.9, 3.1 Hz, 1H),
1H), 6.98 (dd, J ¼ 8.3, 1.6 Hz, 1H), 4.35 (q, J ¼ 6.6, 6.0 Hz, 2H), 3.86 (t,
J ¼ 3.9 Hz, 4H), 3.10 (t, J ¼ 3.4 Hz, 4H), 1.40 (t, J ¼ 7.1 Hz, 3H), 1.33 (s,
3.78 (s, 3H), 3.75 (s, 4H), 3.02 (t, J ¼ 4.6 Hz, 4H); 13C NMR (75 MHz,
DMSO‑d6)
d 166.65, 158.13, 143.39, 126.61, 125.32, 120.39, 114.79,
12H); 13C NMR (75 MHz, CDCl3)
d
168.19, 154.03, 139.08, 138.18,
66.83, 56.03, 53.29; ESI-MS m/z 237.1 [M ꢁ H]- 236.1.
123.21, 117.36, 83.80, 66.91, 60.94, 52.25, 25.01, 24.83, 24.54, 14.37;
Ethyl 5-(benzyloxy)-2-morpholinobenzoate (32b). White
ESI-MS m/z 261.2 [MþH]þ 262.2.
solid, yield 63.3% over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
d 17.42
The synthetic methods of intermediates 29b and 30b were the
same as methods for the preparation of 29a and 30a.
Ethyl 5-bromo-2-(piperidin-1-yl)benzoate (29b). colorless oil,
(s, 1H), 7.69 (d, J ¼ 8.9 Hz, 1H), 7.58 (d, J ¼ 3.1 Hz, 1H), 7.54e7.42 (m,
2H), 7.40 (s, 1H), 7.37 (d, J ¼ 6.0 Hz, 1H), 7.31 (dd, J ¼ 8.2, 5.0 Hz, 2H),
5.17 (s, 2H), 3.78 (t, J ¼ 4.8 Hz, 4H), 3.04 (t, J ¼ 5.0 Hz, 4H); 13C NMR
yield 89.5%. 1H NMR (300 MHz, CDCl3)
d
7.81 (d, J ¼ 2.5 Hz, 1H), 7.48
(75 MHz, DMSO‑d6) d 166.71, 157.12, 143.58, 137.14, 128.93, 128.38,
(dd, J ¼ 8.8, 2.5 Hz, 1H), 6.92 (d, J ¼ 8.8 Hz, 1H), 4.40 (q, J ¼ 7.1 Hz,
128.08, 125.22, 120.98, 115.87, 70.06, 66.83, 53.28; ESI-MS m/z 313.1
2H), 3.09e2.88 (m, 4H), 1.85e1.67 (m, 4H), 1.65e1.55 (m, 2H), 1.43
[M ꢁ H]- 312.1.
(t, J ¼ 7.1 Hz, 3H); 13C NMR (75 MHz, CDCl3)
d
167.02, 152.15, 134.92,
Ethyl 5-methoxy-2-(piperidin-1-yl)benzoate (32c). White
133.75, 125.89, 120.57, 112.75, 61.21, 53.72, 26.04, 24.10, 14.32; ESI-
solid, yield 54.6% over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
d 18.73
MS m/z 311.1 [MþH]þ 312.1.
(s, 1H), 7.69 (dd, J ¼ 8.9, 3.6 Hz, 1H), 7.58e7.47 (m, 1H), 7.22 (d,
Ethyl
2-(piperidin-1-yl)-5-(4,4,5,5-tetramethyl-1,3,2-
J ¼ 8.9 Hz, 1H), 3.80 (s, 3H), 3.04 (t, J ¼ 5.2 Hz, 4H), 1.87e1.69 (m,
dioxaborolan-2-yl)benzoate (30b). colorless oil, yield 85.4%. 1H
NMR (300 MHz, CDCl3)
8.10 (d, J ¼ 1.6 Hz, 1H), 7.81 (dd, J ¼ 8.2,
1.7 Hz,1H), 6.99 (d, J ¼ 8.3 Hz,1H), 4.39 (q, J ¼ 7.1 Hz, 2H), 3.15e3.04
4H), 1.62 (s, 2H); 13C NMR (75 MHz, DMSO‑d6)
d 166.99, 158.36,
d
143.12, 126.76, 124.75, 120.01, 114.52, 55.99, 54.24, 26.02, 22.47;
ESI-MS m/z 235.1 [M ꢁ H]- 234.1.
22

H. Zhu, W. Li, W. Shuai et al.
European Journal of Medicinal Chemistry 216 (2021) 113316
4.1.15. The general procedures for the preparation of intermediates
33a-c
125.61, 123.89, 123.44, 122.60, 114.24, 114.10, 66.82, 55.87, 53.24;
ESI-MS m/z 356.1 [M ꢁ H]- 355.1.
To the solutions of intermediate 26 (200 mg, 0.8 mmol) in 10 mL
CH3OH, various aldehydes (0.96 mmol) were added and stirred for
30 min, then NaBH4 (46 mg, 1.2 mmol) was added and stirred for
another 15 min. The reactions were quenched with saturated NH4Cl
aqueous and extracted with EtOAc (3 ꢃ 25 mL), and the combined
organic layers were washed with saturated brine, dried over
anhydrous Na2SO4, and concentrated in vacuo to afford the crude
products. To the solutions of the above crude products in 10 mL
CH3OH, 10 mL 10% NaOH aqueous was added and the mixtures
were stirred at 80 ꢀC for 30 min CH3OH was then removed in vacuo
and acidized with 10% HCl aqueous to pH 3. The precipitates were
collected by filtration, washed with water and dried to afford 33a-c.
5-(Isonicotinamido)-2-morpholinobenzoic acid (33e). Grey
solid, yield 46.8% over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
d 16.83
(s, 1H), 10.69 (s, 1H), 8.77 (s, 2H), 8.38 (s, 1H), 8.07 (d, J ¼ 8.7 Hz, 1H),
7.85 (s, 2H), 7.69 (d, J ¼ 8.7 Hz, 1H), 3.77 (s, 4H), 3.04 (s, 4H); 13C
NMR (75 MHz, DMSO‑d6)
d 172.36, 166.90, 164.54, 150.80, 146.49,
141.84, 137.37, 125.72, 123.93, 122.88, 122.01, 66.83, 53.24; ESI-MS
m/z 327.1 [M ꢁ H]- 326.1.
5-(Furan-2-carboxamido)-2-morpholinobenzoic acid (33f).
Grey solid, yield 50.9% over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
d
16.84 (s, 1H), 10.34 (s, 1H), 8.31 (d, J ¼ 2.6 Hz, 1H), 7.98 (dd, J ¼ 8.8,
2.7 Hz, 1H), 7.86 (d, J ¼ 1.6 Hz, 1H), 7.59 (d, J ¼ 8.8 Hz, 1H), 7.30 (d,
J ¼ 3.5 Hz, 1H), 6.62 (dd, J ¼ 3.5, 1.7 Hz, 1H), 3.71 (t, 4H), 2.96 (t,
5-((4-Methoxybenzyl)amino)-2-morpholinobenzoic
(33a)Grey solid, yield 66.9% over 2 steps. 1H NMR (300 MHz,
DMSO‑d6)
acid
J ¼ 4.6 Hz, 4H); 13C NMR (75 MHz, DMSO‑d6)
d 166.87, 161.45,
156.75, 147.59, 146.45, 146.05, 137.51, 125.70, 123.98, 122.60, 115.57,
d
7.39 (d, J ¼ 8.5 Hz, 1H), 7.23 (d, J ¼ 8.7 Hz, 2H), 6.85 (d,
112.67, 66.81, 53.19; ESI-MS m/z 316.1 [M ꢁ H]- 315.1.
J ¼ 8.2 Hz, 2H), 6.81e6.75 (m, 1H), 6.61 (d, J ¼ 5.9 Hz, 1H), 4.19 (s,
5-Acetamido-2-morpholinobenzoic acid (33g). White solid,
2H), 3.75e3.70 (m, 4H), 3.68 (s, 3H), 2.93 (t, J ¼ 4.6 Hz, 4H); 13C
yield 64.5% over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
d 10.14 (s,
NMR (75 MHz, DMSO‑d6)
d
167.34, 158.63, 148.08, 138.74, 131.82,
1H), 8.17 (d, J ¼ 2.6 Hz, 1H), 7.86 (dd, J ¼ 8.8, 2.6 Hz, 1H), 7.61 (d,
128.75, 125.64, 124.38, 117.31, 114.25, 113.47, 66.87, 55.46, 53.38,
J ¼ 8.7 Hz,1H), 3.76 (t, J ¼ 4.5 Hz, 4H), 3.00 (t, J ¼ 4.5 Hz, 4H), 2.02 (s,
46.19; ESI-MS m/z 342.2 [M ꢁ H]- 341.2.
3H); 13C NMR (75 MHz, DMSO‑d6)
d 168.99, 166.82, 145.37, 138.34,
2-Morpholino-5-((pyridin-4-ylmethyl)amino)benzoic acid
(33b). Grey solid, yield 54.4% over 2 steps. 1H NMR (300 MHz,
125.70, 124.32, 124.12, 121.16, 66.80, 53.21, 24.40; ESI-MS m/z 264.1
[M ꢁ H]- 263.1.
DMSO‑d6)
d
17.73 (s, 1H), 8.48 (d, J ¼ 5.1 Hz, 2H), 7.44 (d, J ¼ 8.7 Hz,
1H), 7.33 (d, J ¼ 5.2 Hz, 2H), 7.21 (d, J ¼ 2.9 Hz, 1H), 6.82 (s, 1H), 6.78
(dd, J ¼ 8.7, 3.0 Hz, 1H), 4.35 (d, J ¼ 4.1 Hz, 2H), 3.75 (t, 4H), 2.95 (t,
4.1.17. The general procedures for the preparation of intermediates
33h-j
J ¼ 4.4 Hz, 4H); 13C NMR (75 MHz, DMSO‑d6)
d
167.25, 149.99,
To a solution of 30a (200 mg, 0.55 mmol) in 20 mL DMF, various
bromobenzenes (0.66 mmol), Pd(PPh3)4 (60 mg, 0.06 mmol), and
K2CO3 (152 mg, 1.1 mmol) were added. The mixture was stirred at
90 ꢀC for 2 h. Then, the mixtures were filtered and the filtrates were
concentrated in vacuo to afford the crude products, which were
dissolved into 10 mL CH3OH, 10 mL 10% NaOH aqueous was added
and the mixtures were stirred at 80 ꢀC for 30 min CH3OH was then
removed in vacuo and acidized with 10% HCl aqueous to pH 3. The
precipitates were collected by filtration, washed with water and
dried to afford 33h-j.
149.63, 147.69, 139.16, 125.78, 124.57, 122.61, 117.31, 113.53, 66.85,
53.35, 45.69; ESI-MS m/z 313.1 [M ꢁ H]- 312.1.
5-((Furan-2-ylmethyl)amino)-2-morpholinobenzoic
acid
(33c). Grey solid, yield 49.8% over 2 steps. 1H NMR (300 MHz,
DMSO‑d6)
d
7.54 (s, 1H), 7.44 (d, J ¼ 8.7 Hz, 1H), 7.27 (d, J ¼ 2.7 Hz,
1H), 6.96e6.82 (m, 1H), 6.53 (t, J ¼ 6.1 Hz, 1H), 6.35 (s, 1H), 6.25 (d,
J ¼ 3.1 Hz,1H), 4.24 (d, J ¼ 5.9 Hz, 2H), 3.74 (t, J ¼ 4.5 Hz, 4H), 2.95 (t,
J ¼ 4.5 Hz, 4H); 13C NMR (75 MHz, DMSO‑d6)
d 167.30, 153.25,
147.74, 142.54, 139.09, 125.67, 124.43, 117.34, 113.55, 110.84, 107.45,
66.88, 53.38; ESI-MS m/z 302.1 [M ꢁ H]- 301.1.
4′-Methoxy-4-morpholino-[1,1′-biphenyl]-3-carboxylic acid
(33h)White solid, yield 57.9% over 2 steps. 1H NMR (300 MHz,
4.1.16. The general procedures for the preparation of intermediates
33d-g
DMSO‑d6)
d
8.11 (s, 1H), 7.82 (s, 1H), 7.59 (s, 2H), 7.00 (s, 2H), 3.77 (s,
167.40, 166.41,
7H), 3.05 (s, 4H); 13C NMR (75 MHz, DMSO‑d6)
d
To the solutions of intermediate 26 (200 mg, 0.8 mmol) in 10 mL
CH3CN, various benzoic acids (0.96 mmol), HATU (366 mg,
159.64, 149.46, 137.85, 131.46, 128.56, 128.21, 125.88, 123.46, 114.97,
66.80, 55.64, 53.03; ESI-MS m/z 313.1 [M ꢁ H]- 312.1.
0.96 mmol), and Et3N (134
m
L, 0.96 mmol) were added. The mix-
2-Morpholino-5-(pyridin-4-yl)benzoic acid (33i). Grey solid,
tures were stirred for 2 h, and extracted with DCM (3 ꢃ 25 mL). The
combined organic layers were then washed with brine, dried over
anhydrous Na2SO4, and concentrated in vacuo to provide the crude
products. To the solutions of the above products in 10 mL CH3OH,
10 mL 10% NaOH aqueous was added and the mixtures were stirred
at 80 ꢀC for 30 min CH3OH was then removed in vacuo and acidized
with 10% HCl aqueous to pH 3. The precipitates were collected by
filtration, washed with water and dried to afford 33d-f. For inter-
mediate 33g, intermediate 26 (200 mg, 0.8 mmol) in 10 mL CH3CN,
yield 44.3% over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
d
15.71 (s,
1H), 8.10 (s, 2H), 7.89 (d, J ¼ 8.4 Hz, 2H), 7.79e7.38 (m, 3H), 3.77 (s,
4H), 3.07 (s, 6H); 13C NMR (75 MHz, DMSO‑d6)
167.54, 156.64,
d
150.41, 146.73, 135.80, 131.70, 128.98, 125.89, 123.16, 116.74, 66.75,
53.35, 52.88; ESI-MS m/z 282.1 [M ꢁ H]- 281.1.
5-(Furan-2-yl)-2-morpholinobenzoic acid (33j). White solid,
yield 55.4% over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
d 15.93 (s,
1H), 8.23 (d, J ¼ 2.2 Hz, 1H), 7.91 (dd, J ¼ 8.5, 2.3 Hz, 1H), 7.78 (d,
J ¼ 1.7 Hz, 1H), 7.59 (d, J ¼ 8.5 Hz, 1H), 7.03 (d, J ¼ 3.4 Hz, 1H), 6.62
acetic anhydride (91
mL, 0.96 mmol), and Et3N (134
mL, 0.96 mmol)
(dd, J ¼ 3.4, 1.8 Hz, 1H), 3.88e3.69 (m, 4H), 3.16e2.98 (m, 4H); 13
C
were added. The mixtures were stirred for 2 h, and extracted with
EtOAc (3 ꢃ 25 mL). The combined organic layers were then washed
with brine, dried over anhydrous Na2SO4, and concentrated in
vacuo to provide the crude product, which was further hydrolyzed
in above procedure to afford 33g.
NMR (75 MHz, DMSO‑d6) d 167.36, 152.19, 149.77, 143.76, 128.56,
127.92, 125.90, 125.78, 123.13, 112.70, 106.94, 66.71, 52.85; ESI-MS
m/z 273.1 [M ꢁ H]- 272.1.
4.1.18. The general procedures for the preparation of target
compounds 34a-j
The synthetic methods of 34a-j were the same as method B for
the preparation of 20a-p.
5-(4-Methoxybenzamido)-2-morpholinobenzoic acid (33d).
White solid, yield 42.5% over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
d
10.30 (s,1H), 8.39 (s,1H), 8.09 (d, J ¼ 8.7 Hz,1H), 7.96 (d, J ¼ 8.4 Hz,
1H), 7.86 (d, J ¼ 8.3 Hz, 2H), 7.04 (d, J ¼ 8.3 Hz, 1H), 6.98 (d,
N-(3-hydroxy-4-methoxybenzyl)-5-((4-methoxybenzyl)
amino)-2-morpholinobe nzamide (34a). White solid, yield 66.9%
J ¼ 8.4 Hz, 2H), 3.81 (s, 3H), 3.79 (s, 4H), 3.03 (s, 4H); 13C NMR
(75 MHz, DMSO‑d6)
d
167.44, 163.27, 138.41, 138.41, 131.78, 130.17,
over 2 steps. 1H NMR (300 MHz, CDCl3)
d
10.84 (d, J ¼ 5.4 Hz, 1H),
23

H. Zhu, W. Li, W. Shuai et al.
European Journal of Medicinal Chemistry 216 (2021) 113316
7.62 (d, J ¼ 3.0 Hz, 1H), 7.32e7.25 (m, 2H), 7.05 (d, J ¼ 8.6 Hz, 1H),
6.96 (d, J ¼ 1.9 Hz, 1H), 6.92e6.74 (m, 4H), 6.65 (dd, J ¼ 8.5, 3.0 Hz,
1H), 5.95 (s, 1H), 4.52 (d, J ¼ 5.1 Hz, 2H), 4.27 (s, 2H), 3.87 (s, 3H),
3.79 (s, 3H), 3.46 (s, 4H), 2.79 (d, J ¼ 4.7 Hz, 4H); 13C NMR (75 MHz,
452.1816 found 452.1817.
5-Acetamido-N-(3-hydroxy-4-methoxybenzyl)-2-
morpholinobenzamide (34g). White solid, yield 43.3% over 2
steps. 1H NMR (300 MHz, DMSO‑d6)
d 10.02 (s, 1H), 9.89 (d,
CDCl3)
d
165.92, 158.94, 146.27, 146.08, 146.00, 140.98, 131.80,
J ¼ 5.9 Hz, 1H), 8.96 (s, 1H), 7.98 (s, 1H), 7.77 (d, J ¼ 8.6 Hz, 1H), 7.22
(d, J ¼ 8.7 Hz, 1H), 6.87 (d, J ¼ 8.2 Hz, 1H), 6.82 (s, 1H), 6.76 (d,
J ¼ 8.1 Hz, 1H), 4.36 (s, 2H), 3.74 (s, 3H), 3.45 (t, J ¼ 4.2 Hz, 4H),
2.96e2.67 (m, 4H), 2.03 (s, 3H); 13C NMR (75 MHz, DMSO‑d6)
131.19, 128.88, 128.44, 122.73, 119.99, 115.62, 115.55, 114.72, 114.13,
110.86, 67.08, 56.12, 55.39, 53.75, 47.87, 43.64; HR-MS (ESI) m/z:
calcd for C27H32N3O5 [MþH]þ 478.2336 found 478.2333.
N-(3-hydroxy-4-methoxybenzyl)-2-morpholino-5-((pyridin-
4-ylmethyl)amino) benzamide (34b). White solid, yield 50.2%
d 168.22, 165.34, 146.79, 146.57, 145.74, 135.73, 131.66, 128.82,
121.99, 121.18, 120.76, 118.68, 115.33, 112.22, 66.04, 55.73, 52.92,
42.38, 23.91; HR-MS (ESI) m/z: calcd for C21H26N3O5 [MþH]þ
400.1867 found 400.1868.
over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
d
10.45 (d, J ¼ 6.3 Hz,
1H), 8.95 (d, J ¼ 3.0 Hz, 1H), 8.56e8.42 (m, 2H), 7.38e7.29 (m, 2H),
7.24 (d, J ¼ 2.9 Hz,1H), 7.10 (d, J ¼ 8.6 Hz,1H), 6.86 (d, J ¼ 8.2 Hz,1H),
6.79 (s,1H), 6.74 (dd, J ¼ 8.1, 2.1 Hz,1H), 6.62 (dd, J ¼ 8.7, 3.0 Hz,1H),
6.53 (d, J ¼ 6.9 Hz, 1H), 4.39e4.26 (m, 4H), 3.73 (s, 3H), 3.40 (dd,
J ¼ 5.9, 3.2 Hz, 4H), 2.70 (t, J ¼ 4.6 Hz, 4H); 13C NMR (75 MHz,
N-(3-hydroxy-4-methoxybenzyl)-4′-methoxy-4-
morpholino-[1,1′-biphenyl]-3-carboxamide (34h). White solid,
yield 57.9% over 2 steps. 1H NMR (300 MHz, CDCl3)
d 10.06 (d,
J ¼ 4.8 Hz, 1H), 8.44 (d, J ¼ 2.4 Hz, 1H), 7.62 (dd, J ¼ 8.3, 2.5 Hz, 1H),
7.60e7.51 (m, 2H), 7.22 (d, J ¼ 8.3 Hz, 1H), 7.05e6.90 (m, 3H), 6.88
(dd, J ¼ 8.2, 2.0 Hz, 1H), 6.83 (d, J ¼ 8.2 Hz, 1H), 5.88 (s, 1H), 4.57 (d,
J ¼ 5.3 Hz, 2H), 3.88 (s, 3H), 3.85 (s, 3H), 3.52 (t, J ¼ 4.4 Hz, 4H), 2.92
DMSO‑d6)
d 165.23, 149.56, 146.83, 146.59, 146.45, 140.22, 131.66,
128.78, 122.71, 122.18, 118.62, 115.27, 115.17, 114.83, 113.92, 112.23,
66.22, 55.71, 53.28, 45.47, 42.37; HR-MS (ESI) m/z: calcd for
C
25H29N4O4 [MþH]þ 449.2183 found 449.2184.
(t, J ¼ 4.6 Hz, 4H); 13C NMR (75 MHz, CDCl3)
d 166.00,159.37,149.36,
5-((Furan-2-ylmethyl)amino)-N-(3-hydroxy-4-
146.31, 146.10, 137.67, 132.33, 131.68, 129.95, 129.89, 128.09, 127.98,
121.00, 120.04, 114.68, 114.35, 110.90, 66.94, 56.14, 55.46, 53.54,
43.64; HR-MS (ESI) m/z: calcd for C26H29N2O5 [MþH]þ 449.2071
found 449.2069.
methoxybenzyl)-2-morpholinobe nzamide (34c). White solid,
yield 54.2% over 2 steps. 1H NMR (300 MHz, CDCl3)
10.84 (t,
d
J ¼ 5.3 Hz,1H), 7.63 (d, J ¼ 3.0 Hz,1H), 7.35 (d, J ¼ 1.8 Hz,1H), 7.08 (d,
J ¼ 8.6 Hz, 1H), 6.95 (d, J ¼ 1.9 Hz, 1H), 6.88e6.80 (m, 2H), 6.73 (dd,
J ¼ 8.6, 3.0 Hz, 1H), 6.31 (dd, J ¼ 3.2, 1.8 Hz, 1H), 6.25 (d, J ¼ 3.2 Hz,
1H), 6.01 (s, 1H), 4.52 (d, J ¼ 5.2 Hz, 2H), 4.33 (s, 2H), 4.23 (s, 1H),
3.88 (s, 3H), 3.45 (d, J ¼ 4.8 Hz, 4H), 2.80 (t, J ¼ 4.6 Hz, 4H); 13C NMR
N-(3-hydroxy-4-methoxybenzyl)-2-morpholino-5-(pyridin-
4-yl)benzamide (34i). White solid, yield 53.4% over 2 steps. 1H
NMR (300 MHz, CDCl3)
d
9.74 (t, J ¼ 5.4 Hz, 1H), 8.66 (d, J ¼ 5.1 Hz,
2H), 8.51 (d, J ¼ 2.3 Hz, 1H), 7.73 (dd, J ¼ 8.4, 2.3 Hz, 1H), 7.55 (d,
J ¼ 5.1 Hz, 2H), 7.30 (s, 1H), 6.98 (s, 1H), 6.86 (q, J ¼ 8.3 Hz, 2H), 6.15
(s, 1H), 4.58 (d, J ¼ 5.3 Hz, 2H), 3.90 (s, 3H), 3.55 (t, J ¼ 4.4 Hz, 4H),
(75 MHz, CDCl3)
d 165.84, 152.46, 146.28, 146.07, 145.41, 142.04,
141.45, 131.70, 128.38, 122.70, 119.94, 116.10, 115.65, 114.66, 110.88,
110.43, 107.26, 67.02, 56.11, 53.70, 43.59, 41.45; HR-MS (ESI) m/z:
calcd for C24H28N3O5 [MþH]þ 438.2023 found 438.2022.
2.96 (t, J ¼ 4.5 Hz, 4H); 13C NMR (75 MHz, CDCl3)
d 165.61, 151.52,
150.24, 147.11, 146.52, 146.28, 134.44, 131.44, 130.54, 130.32, 128.47,
121.46, 121.01, 119.98, 114.75, 110.99, 66.81, 56.14, 53.44, 43.68; HR-
MS (ESI) m/z: calcd for C23H26N3O4 [MþH]þ 420.1918 found
420.1910.
N-(3-hydroxy-4-methoxybenzyl)-5-(4-methoxybenzamido)-
2-morpholinobenza mide (34d). White solid, yield 46.8% over 2
steps. 1H NMR (300 MHz, CDCl3)
d 10.49 (s, 1H), 8.48 (s, 1H),
8.41e8.34 (m, 1H), 8.09 (s, 1H), 7.89 (d, J ¼ 8.5 Hz, 2H), 7.23 (s, 1H),
5-(Furan-2-yl)-N-(3-hydroxy-4-methoxybenzyl)-2-
morpholinobenzamide (34j). White solid, yield 56.9% over 2 steps.
6.96 (s, 1H), 6.93 (s, 2H), 6.81 (d, J ¼ 1.2 Hz, 2H), 5.94 (s, 1H), 4.46 (d,
J ¼ 5.2 Hz, 2H), 3.89 (s, 3H), 3.85 (s, 3H), 3.49 (s, 4H), 2.88 (s, 4H); 13
C
1H NMR (300 MHz, CDCl3)
d
9.94 (s, 1H), 8.49 (d, J ¼ 2.3 Hz,1H), 7.75
NMR (75 MHz, DMSO‑d6)
d
165.48, 164.71, 161.93, 146.84, 146.58,
(dd, J ¼ 8.4, 2.4 Hz, 1H), 7.47 (d, J ¼ 1.8 Hz, 1H), 7.20 (d, J ¼ 8.4 Hz,
1H), 6.97 (d, J ¼ 2.1 Hz, 1H), 6.94e6.78 (m, 2H), 6.71 (d, J ¼ 3.4 Hz,
1H), 6.48 (dd, J ¼ 3.5, 1.8 Hz, 1H), 5.74 (s, 1H), 4.57 (d, J ¼ 5.4 Hz, 2H),
3.90 (s, 3H), 3.66e3.39 (m, 4H), 2.92 (t, J ¼ 4.6 Hz, 4H); 13C NMR
146.08, 135.66, 131.70, 129.61, 128.76, 126.70, 123.24, 122.24, 120.86,
118.65, 115.35, 113.60, 112.22, 66.04, 55.72, 55.44, 52.91, 42.38; HR-
MS (ESI) m/z: calcd for C27H30N3O6 [MþH]þ 492.2129 found
492.2126.
(75 MHz, CDCl3)
d 165.75, 153.01, 149.58, 146.33, 146.12, 142.31,
N-(3-((3-hydroxy-4-methoxybenzyl)carbamoyl)-4-
morpholinophenyl)isonicotin amide (34e). White solid, yield
131.56, 128.03, 128.01, 127.34, 127.03, 120.84, 120.02, 114.69, 111.89,
110.92,105.59, 66.86, 56.12, 53.44, 43.64; HR-MS (ESI) m/z: calcd for
44.4% over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
d
10.57 (s, 1H),
C
23H25N2O5 [MþH]þ 409.1758 found 409.1752.
9.77 (t, J ¼ 5.8 Hz, 1H), 8.94 (s, 1H), 8.83e8.70 (m, 2H), 8.19 (d,
J ¼ 2.6 Hz, 1H), 7.95 (dd, J ¼ 8.7, 2.7 Hz, 1H), 7.92e7.79 (m, 2H), 7.29
(d, J ¼ 8.8 Hz, 1H), 6.88 (d, J ¼ 8.2 Hz, 1H), 6.83 (d, J ¼ 2.0 Hz, 1H),
6.77 (dd, J ¼ 8.2, 2.0 Hz, 1H), 4.38 (d, J ¼ 5.7 Hz, 2H), 3.74 (s, 3H),
3.48 (t, J ¼ 4.2 Hz, 4H), 2.83 (t, J ¼ 4.5 Hz, 4H); 13C NMR (75 MHz,
4.1.19. 5-((5-fluoro-2-morpholinobenzamido)methyl)-2-
methoxyphenyl phosphate disodium (20b-P)
To a solution of 20b (1.0 g, 2.77 mmol) in anhydrous THF, NaH
(133 mg, 3.33 mmol, 60%) was added and stirred at 0 ꢀC under
nitrogen atmosphere for 15 min. Then, newly prepared dibenzyl-
phosphoryl chloride (988 mg, 3.33 mmol) was added, and the
mixture was stirred at room temperature for 2 h. The mixture was
extracted with EtOAc (3 ꢃ 25 mL), and the combined organic layers
were washed with saturated brine, dried over anhydrous Na2SO4,
and concentrated in vacuo to afford the crude product, which was
purified by column chromatography with petroleum/ethyl acetate
(5:1) to give 1.1 g 20b-Bn as white solid, yield 63.8%. 20b-Bn (1.1 g,
2.77 mmol) was dissolved into 20 mL CH3OH, PdeC (100 mg) was
added. The reaction was stirred for 2 h under hydrogen atmo-
sphere, then the mixture was filtered and the filtrate was concen-
trated in vacuo to provide 750 mg crude product as white solid,
which was dissolved into 20 mL CH3OH, and NaOH (129 mg,
3.24 mmol) was added. After stirring for 30 min, the mixture was
DMSO‑d6)
d 165.42, 163.74, 150.30, 146.84, 146.72, 146.56, 141.65,
134.74, 131.67, 128.91, 123.34, 122.41, 121.57, 120.88, 118.67, 115.35,
112.20, 66.00, 55.72, 52.82, 42.38; HR-MS (ESI) m/z: calcd for
C
25H27N4O5 [MþH]þ 463.1976 found 463.1970.
N-(3-((3-hydroxy-4-methoxybenzyl)carbamoyl)-4-
morpholinophenyl)furan-2-carboxamide (34f). White solid,
yield 42.9% over 2 steps. 1H NMR (300 MHz, CDCl3)
d
10.35 (t,
J ¼ 5.4 Hz,1H), 8.32 (m, 2H), 8.02 (d, J ¼ 2.8 Hz,1H), 7.53 (s, 1H), 7.24
(d, J ¼ 4.4 Hz, 1H), 6.97 (s, 1H), 6.83 (t, J ¼ 6.1 Hz, 2H), 6.56 (s, 1H),
5.89 (s, 1H), 4.55 (d, J ¼ 5.2 Hz, 2H), 3.89 (s, 3H), 3.55e3.44 (m, 4H),
2.89 (t, J ¼ 4.5 Hz, 4H); 13C NMR (75 MHz, CDCl3)
d 165.20, 156.35,
147.70, 147.04, 146.43, 146.19, 144.60, 135.07, 131.47, 128.33, 123.87,
122.73, 122.19, 120.02, 115.56, 114.75, 112.71, 110.96, 66.91, 56.16,
53.62, 43.72; HR-MS (ESI) m/z: calcd for C24H25N2O6 [MþH]þ
24

H. Zhu, W. Li, W. Shuai et al.
European Journal of Medicinal Chemistry 216 (2021) 113316
concentrated in vacuo. The residue was solidified and washed with
EtOAc to afford 769 mg 20b-P as white solid, yield 89.6% over 2
preincubated with compounds or vehicle DMSO on ice. PEG con-
taining GTP was added to the final concentration of 3 mg/mL before
detecting the tubulin polymerization reaction. After 30 min, the
absorbance was detected by a spectrophotometer at 340 nm at
37 ꢀC every 2 min for 60 min. The area under the curve was used to
determine the concentration that inhibited tubulin polymerization
by 50% (IC50), which was calculated with GraphPad Prism Software
version 5.02.
steps. 1H NMR (300 MHz, MeOD)
d
7.71 (d, J ¼ 1.7 Hz, 1H), 7.62 (dd,
J ¼ 9.5, 3.1 Hz, 1H), 7.32 (dd, J ¼ 8.9, 4.9 Hz, 1H), 7.20 (ddd, J ¼ 8.9,
7.7, 3.2 Hz, 1H), 6.97e6.91 (m, 2H), 4.51 (s, 2H), 3.85 (s, 3H),
3.53e3.46 (m, 4H), 2.89e2.84 (m, 4H); 13C NMR (75 MHz, MeOD)
d
165.83, 161.09, 157.87, 149.52 (d, J ¼ 3.0 Hz), 147.29, 144.43, 130.37
(d, J ¼ 17.3 Hz), 122.77 (d, J ¼ 7.5 Hz), 120.71, 119.97, 118.07 (d,
J ¼ 21.8 Hz),116.45 (d, J ¼ 24.0 Hz), 111.51, 66.49, 55.23, 53.09, 43.24.
HR-MS (ESI) m/z: calcd for C19H21FN2Na2O7P [M ꢁ Na]- 461.0895
found 461.0861.
4.2.5. Competitive inhibition assays
The competitive binding activity of inhibitors was evaluated
using
proximity (SPA) assay. In brief, 0.08
with 20b (1 M, 5 M) or CA-4 (1
porcine tubulin (0.5 g) in a buffer of 100
PIPES (pH 6.8), 1 mM EGTA, 10% glycerol, 1 mM MgCl2, and 1 mM
GTP for 2 h at 37 ꢀC. Then streptavidin-labeled SPA beads (80
g)
a
radiolabeled [3H]-colchicine competition scintillation
M [3H]-colchicine was mixed
M, 5 M) and biotinylated
L containing 80 mM
4.2. Biology
m
m
m
m
m
4.2.1. Materials
m
m
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2-H-tetrazolium
bromide (MTT), propidium iodide (PI), Hoechst 33342, the Annexin
V-FITC apoptosis detection kit, 5,50,6,60-tetrachloro-1,10,3,30-tet-
raethyl-imidacarbocyanine iodide (JC-1) were purchased from
Nanjing KeyGen Biotech Co. Ltd. (Nanjing, China). Goat antimouse
IgG/Alexa-Fluor 488 antibody was purchased from Jackson Immu-
noResearch in USA. Primary antibodies against cdc2, cyclin B1, p-
histone-H3, Bax, Bcl-2, caspase 3, caspase 9, p53, and PARP were
purchased from Beyotime (Jiangsu, China). The purified tubulin
polymerization kit was purchased from Cytoskeleton Inc. (Denver,
USA). Rat plasma and Enalapril were purchased from Tripod
(Jiangsu, China). Other reagents were purchased from Sigma-
Aldrich (St. Louis, MO, USA) unless otherwise specified.
m
were added to each mixture. The radioactive counts were measured
directly with a scintillation counter.
4.2.6. Immunofluorescent staining
A431 cells were seeded into 6-well plates and then treated with
vehicle control 0.1% DMSO, and 20b (7.5 nM, 15 nM, 30 nM). The
cells were fixed with 4% paraformaldehyde and then washed three
times with PBS. After blocking for 20 min by adding 50e100
serum albumin at room temperature, cells were incubated with a
monoclonal antibody (anti-
-tubulin) at 37 ꢀC for 2 h. Then the cells
mL goat
a
were washed three times by PBS following staining by fluorescence
secondary antibody and labeling of nuclei by 4,6-diamidino-2-
phenylindole (DAPI). Cells were visualized on a Zeiss LSM 570
laser scanning confocal microscope (Carl Zeiss, Germany).
4.2.2. Cell lines and cell culture
Human chronic myelogenous leukemia cells (K562), hepato-
cellular carcinoma cells (HepG2), human colon cancer cells (HCT-8),
and human breast cancer cells (MDA-MB-231), mouse liver cancer
cells (H22) were grown in RPMI 1640 medium (KeyGen Biotech Co.
Ltd., Nanjing, China). Human umbilical vein endothelial cells
(HUVECs) and human normal hepatocytes LO2 cells were grown in
F12K medium (KeyGen Biotech Co. Ltd., Nanjing, China). The me-
dium for all cell lines were supplemented with 10% fetal bovine
4.2.7. Molecular modeling
The 2D structures of 20b, IMB5046, and CA-4 were generated on
ChemBioDraw Ultra 12.0. Ligprep module implemented in
€
Schrodinger was used to generate energy minimized 3D structures.
The X-ray structure of CA-4-a,b-tubulin complex was downloaded
from the Protein Data Bank (PDB code 5lyj) and prepared for
docking using Protein Preparation Wizard. Default settings were
used for all the remaining parameters and no constraints were
specified separately. All ligand conformers were docked to each of
the receptor grid structures using Glide extra precision (XP) mode.
The 2D interactions between ligands and tubulin were generated by
Ligand Interaction Diagram module, and the structural image was
obtained using PyMOL software.
serum (Life Technologies, USA), 100
mg/mL streptomycin (Life
Technologies, USA), and 100 U/mL penicillin (Life Technologies,
USA) and maintained at 37 ꢀC in a humidified atmosphere with 5%
CO2.
4.2.3. MTT assay
The overall growth of human cancer cell lines was determined
using the colorimetric MTT assay. Briefly, the cell lines were incu-
bated at 37 ꢀC in a humidified 5% CO2 incubator for 24 h in 96-well
plates prior to the experiments. K562, HepG2, H22, HCT-8, MDA-
MB-231 and L-O2 cells were seeded at a density of 5 ꢃ 103 cells/
4.2.8. Cell cycle analysis
A431 cells were seeded into 6-well plates and incubated at 37 ꢀC
in a humidified 5% CO2 incubator for 24 h, and then treated with or
without 20 b at indicated concentrations (7.5 nM,15 nM, 30 nM) for
another 48 h. The collected cells were fixed by adding 70% ethanol
at 4 ꢀC for 12 h. Subsequently, the cells were resuspended in PBS
containing 100 mL RNase A and 400 mL of propidium iodide for
30 min. The DNA content of the cells was measured using a FACS
Calibur flow cytometer (Bectone Dickinson, San Jose, CA, USA).
well. After removal of medium, 100 mL of fresh medium containing
the test compounds at different concentrations was added to each
well and incubated at 37 ꢀC for 72 h (or 6 h). The percentage of
DMSO in the medium did not exceeded 0.25%. The number of living
cells after 72 h (or 6 h) of culture in the presence (or absence, as in
the case of the control) of the various compounds was directly
proportional to the intensity of the blue color of media, which is
quantitatively measured by spectrophotometry (Biorad, Nazareth,
Belgium) at a 570 nm wavelength. The experiment was performed
in quadruplicate and repeated three times.
4.2.9. Hoechst 33342 staining
Approximately 5 ꢃ 104 cells per well were plated in six-well
plates, and the cells were then incubated with 20b (7.5 nM,
15 nM, 30 nM) for 48 h. After incubation, cells were washed with
PBS, fixed in 4% paraformaldehyde for 30 min, and then stained
4.2.4. In vitro tubulin polymerization inhibitory assay
The tubulin reaction mix in 100 mL PEM buffer contained 2 mg/
mL tubulin (Cytoskeleton), 80 mM piperazine-N,N0-bis(2-
ethanesulfonic acid) sequisodium salt PIPES (pH 6.9), 0.5 mM
EGTA, 2 mM MgCl2, and 15% glycerol. Then, the mixture was
with 20 mg/mL Hoechst 33342 for 15 min at room temperature in
the dark. Cells were then assessed by fluorescence microscopy for
morphological changes after 20b treatment.
25

H. Zhu, W. Li, W. Shuai et al.
European Journal of Medicinal Chemistry 216 (2021) 113316
4.2.10. Cell apoptosis analysis
4.2.16. Determination of acute toxicity
After treatment with or without 20 b at indicated concentra-
tions (7.5 nM,15 nM, 30 nM) for 48 h, A431 cells were washed twice
in PBS, centrifuged and resuspended in 500 mL AnnexinV binding
buffer. The cells were then harvested, washed and stained with
5 mL Annexin V-APC and 5 mL 7-AAD in the dark for 15 min.
Apoptosis was analyzed using a FACS Calibur flow cytometer
(Bectone Dickinson, San Jose, CA, USA).
Five-week-old male Institute of Cancer Research (ICR) mice
were purchased from Shanghai SLAC Laboratory Animals Co. Ltd.
An acute toxicity study by intravenous injection was conducted
according to the guidelines of the Organization for Economic Co-
operation and Development. The animals were weighed and
divided at random into five groups of ten animals. Then, the mice
were intravenously injected with 20b-P (327.7, 409.6, 512.0, 640,
and 800 mg/kg) in saline solutions. The animals were observed
continuously for 14 days.
4.2.11. Mitochondrial membrane potential analysis
After treatment with vehicle control 0.1% DMSO, and 20b
(7.5 nM, 15 nM, 30 nM) for 48 h, the cells were washed in PBS and
4.2.17. Evaluation of in vivo anti-tumor effect
resuspended in 500
m
L JC-1 incubation buffer at 37 ꢀC for 15 min.
A total of 1 ꢃ 106 H22 cells were subcutaneously inoculated into
the right flank of ICR mice according to protocols of tumor trans-
plant research. After incubation for one day, mice were weighted
and divided at random into eight groups with eight animals per
groups. The groups treated with 20b, IMB5046, and CA-4 were
administered in a vehicle of 10% DMF/2% Tween 80/88% saline,
respectively. The groups treated with 20b-P and CA-4P were
administered 20 mg/kg in a vehicle of saline. The positive control
group was treated with PTX (6 mg/kg) by intravenous injection. The
negative control group received a vehicle of 10% DMF/2% Tween 80/
88% saline through intravenous injection. Treatments of 20b, 20b-P,
IMB5046, and CA-4 were conducted at a frequency of intravenous
injection one dose per day for a total 21 consecutive days while the
positive group was treated with PTX at a dose of 6 mg/kg every
other day. Body weights and tumor volumes were measured every
2 days. After the treatments, all the mice were sacrificed and
weighed. The following formula was used to determine tumor
volumes: tumor volume ¼ L ꢃ W2/2, where L is the length and W is
the width. Ratio of inhibition of tumor (%) ¼ (1 ꢁ average tumor
weight of treated group/average tumor weight of control
group) ꢃ 100%. All procedures were performed following institu-
tional approval in accordance with the NIH Guide for the Care and
Use of Laboratory Animals.
The percentage of cells with healthy or collapsed mitochondrial
membrane potentials was monitored by flow cytometry analysis
(Bectone-Dickinson, San Jose, CA, USA).
4.2.12. Measurement of intracellular ROS generation
Intracellular ROS production was detected by using the
peroxide-sensitive fluorescent probe DCF-DA. In brief, after treat-
ment with 20b (7.5 nM, 15 nM, 30 nM) for 48 h, A431 cells were
incubated with 10 mM DCF-DA at 37 ꢀC for 15 min. The intracellular
ROS mediated oxidation of DCF-DA to the fluorescent compound
20,70-dichlorofluorescein (DCF). Then cells were harvested, and the
pellets were suspended in 1 mL PBS. Samples were analyzed at an
excitation wavelength of 480 nm and an emission wavelength of
525 nm by flow cytometry on a FC500 cytometer (Beckman
Coulter).
4.2.13. Solubility evaluation
Solubility was measured separately at pH 7.4 and pH 2.0 by
using an HPLCꢁUV method. Compounds 20b and 20d were added
to phosphate buffer (pH 7.4) or HCl aqueous (pH 2.0) at 20 ꢀC. After
shaking and centrifuging, the supernatant was taken to determine
the concentration of compounds 20b and 20d in each solvent for
calculation of the corresponding solubility. The mobile phase was
menthol-water (80/20, v/v) at a flow rate of 1.0 mL/min with the
detection wavelength at 254 nm. This experiment was repeated in
triplicates.
4.2.18. Wound healing assays
HUVEC cells were grown in 6-well plates for 24 h. Scratches
were made in confluent monolayers using 200
mL pipette tips.
Then, wounds were washed twice with PBS to remove non-
adherent cell debris. Media containing different concentrations of
20b (7.5 nM, 15 nM, 30 nM) were added to the petri dishes. Cells
which migrated across the wound area were photographed using
phase contrast microscopy at 0 h and 24 h.
4.2.14. log P measurement
The DMSO stock solution (10 mg/mL, 20 mL) of 20b, 20d and CA-
4 were added into the mixture solvents of n-octane (1 mL) and
water (1 mL). The mixtures were stirred at room temperature for
24 h and then stood overnight. Each solution (0.5 mL) was trans-
ferred from two phases respectively into other vials for HPLC
analysis. The instrument and conditions were the same as those for
solubility determination. The log P was calculated by the peak area
ratios in n-octane and in water.
4.2.19. Tube formation assay
EC Matrigel matrix was thawed at 4 ꢀC overnight, and HUVECs
suspended in F12K were seeded in 96-well culture plates at a cell
density of 50,000 cells/well after polymerization of the Matrigel at
37 ꢀC for 30 min. They were then treated with 20
mL different
4.2.15. Plasma stability measurement
concentrations of 20b or vehicle for 6 h at 37 ꢀC. Then, the
morphological changes of the cells and tubes formed were
observed and photographed under inverted microscope (OLYMPUS,
Japan).
The stability of compounds 20b and 20d in rat plasma was
determined using HPLC method. Rat blood plasma was diluted with
distilled water until the indicated volume, and then the solution
was pre-incubated for 5 min at 37 ꢀC. The stock solutions of the test
compounds 20b, 20d and positive control (100 nM, 20
added to the rat plasma (480 L), and the mixtures were incubated
at 37 ꢀC. The incubations were terminated at 0, 15, 30, 60, 120, and
180 min, by removing aliquots (80 L) of the plasma samples and
m
L) were
4.2.20. Immunohistochemistry assay
Slides from mice tissues (control group and 20b-treated group
at 20 mg/kg in in vivo antitumor experiments) were embedded in
m
m
paraffin and cut to sections of 4 mm, deparaffinized, and treated
mixing them with an equal volume of acetonitrile. The mixture was
stirred vigorously and centrifuged (5500 rpm, 5 min). The super-
natant was filtered, and the filtrate was analyzed by HPLC. These
tests were conducted three times for each compound. Remaining
(%) was calculate by the ratio of peak area at each time point and
peak area at 0 min.
with citrate buffer. Then, they were blocked with avidin/biotin for
20 min. The slides were incubated with CD31 overnight at 4 ꢀC.
Next, the slides were treated with secondary antibody with
horseradish peroxidase goat anti-rabbit for 1e3 h and developed
with 3, 3-diaminobenzidine. Finally, the slides were counterstained
with hematoxylin. The mean microvessel density (MVD) was
26

H. Zhu, W. Li, W. Shuai et al.
European Journal of Medicinal Chemistry 216 (2021) 113316
A.P. Piccionello, A. Montalbano, S. Alcaro, E. Hamel, G. Viola, P. Barraja, Insight
on [1,3]thiazolo[4,5-e]isoindoles as tubulin polymerization inhibitors, Eur. J.
Med. Chem. 212 (2021) 113122.
measured by calculating the CD31-positive cells in randomly
selected areas in each section using image analysis software and
then analyzed with OriginPro 8.0 software.
ꢁ
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[19] M.J. Perez-Perez, E.M. Priego, O. Bueno, M.S. Martins, M.D. Canela, S. Liekens,
Blocking blood flow to solid tumors by destabilizing tubulin: an approach to
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[21] C.M. Lin, S.B. Singh, P.S. Chu, R.O. Dempcy, J.M. Schmidt, G.R. Pettit, E. Hamel,
Interactions of tubulin with potent natural and synthetic analogs of the
antimitotic agent combretastatin: a structure-activity study, Mol. Pharmacol.
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Declaration of competing interest
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.
[22] K. Grosios, S.E. Holwell, A.T. McGown, G.R. Pettit, M.C. Bibby, In vivo and
in vitro evaluation of combretastatin A-4 and its sodium phosphate prodrug,
Br. J. Cancer 81 (1999) 1318e1327.
Acknowledgments
[23] G.M. Tozer, V.E. Prise, J. Wilson, R.J. Locke, B. Vojnovic, M.R. Stratford,
M.F. Dennis, D.J. Chaplin, Combretastatin A-4 phosphate as a tumor vascular-
targeting agent: early effects in tumors and normal tissues, Cancer Res 59
(1999) 1626e1634.
[24] http://investor.mateon.com/releasedetail.cfm?ReleaseID¼1041745.
[25] N. Sirisoma, A. Pervin, H. Zhang, S. Jiang, J.A. Willardsen, M.B. Anderson,
G. Mather, C.M. Pleiman, S. Kasibhatla, B. Tseng, J. Drewe, S.X. Cai, Discovery of
This study was supported from the National Natural Science
Foundation of China (No. 81673306, 81703348, 81874289 and
81973167) for financial support, and “Double First-Class” University
project CPU2018GY04, CPU2018GY35, China Pharmaceutical
University.
N-(4-methoxyphenyl)-N,2-dimethylquinazolin-4-amine,
a potent apoptosis
inducer and efficacious anticancer agent with high blood brain barrier
penetration, J. Med. Chem. 52 (2009) 2341e2351.
Appendix A. Supplementary data
[26] K. Mahal, M. Resch, R. Ficner, R. Schobert, B. Biersack, T. Mueller, Effects of the
tumor-vasculature-disrupting agent verubulin and two heteroaryl analogues
on cancer cells, endothelial cells, and blood vessels, ChemMedChem 9 (2014)
847e854.
Supplementary data to this article can be found online at
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[27] X.F. Wang, F. Guan, E. Ohkoshi, W. Guo, L. Wang, D.Q. Zhu, S.B. Wang,
L.T. Wang, E. Hamel, D. Yang, L. Li, K. Qian, S.L. Morris-Natschke, S. Yuan,
K.H. Lee, L. Xie, Optimization of 4-(N-cycloamino)phenylquinazolines as a
novel class of tubulin-polymerization inhibitors targeting the colchicine site,
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