Design, synthesis and biological evaluation of novel 3-substituted 4-anilino-coumarin derivatives as antitumor agents

Article abstract of DOI:10.1016/j.bmcl.2017.01.013

Various 3-substituted 4-anilino-coumarin derivatives have been designed, synthesized and their anti-proliferative properties have been studied. The in vitro cytotoxicity screening was performed against MCF-7, HepG2, HCT116 and Panc-1 cancer cell lines by MTT assay. Most of the synthesized compounds exhibited comparable anti-proliferative activity to the positive control 5-Fluorouracil against these four tested cancer cell lines. Among the different substituents at C-3 position of coumarin scaffold, 3-trifluoroacetyl group showed the most promising results. Especially, compounds 33d (IC₅₀?=?16.57, 5.45, 4.42 and 5.16?μM) and 33e (IC₅₀?=?20.14, 6.71, 4.62 and 5.62?μM) showed excellent anti-proliferative activities on MCF-7, HepG2, HCT116 and Panc-1 cell lines respectively. In addition, cell cycle analysis and apoptosis activation revealed that 33d induced G2/M phase arrest and apoptosis in MCF-7 cells in a dose-dependent manner. Low toxicity of compounds 33d and 33e was observed against human umbilical vein endothelial cells (HUVECs), suggesting their acceptable safety profiles in normal cells. Furthermore, the results of in silico ADME studies indicated that both 33d and 33e exhibited good pharmacokinetic properties.

Full text of DOI:10.1016/j.bmcl.2017.01.013

Accepted Manuscript
Design, synthesis and biological evaluation of novel 3-substituted 4-anilino-
coumarin derivatives as antitumor agents
Guoshun Luo, Moses Muyaba, Weiting Lyu, Zhichao Tang, Ruheng Zhao, Qian
Xu, Qidong You, Hua Xiang
PII:
S0960-894X(17)30013-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.01.013
BMCL 24589
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 October 2016
4 January 2017
6 January 2017
Please cite this article as: Luo, G., Muyaba, M., Lyu, W., Tang, Z., Zhao, R., Xu, Q., You, Q., Xiang, H., Design,
synthesis and biological evaluation of novel 3-substituted 4-anilino-coumarin derivatives as antitumor agents,
Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.01.013
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Design, synthesis and biological evaluation of novel 3-substituted 4-anilino-coumarin
derivatives as antitumor agents
b
Guoshun Luo a, b, Moses Muyaba a, b, Weiting Lyu , Zhichao Tang a, b, Ruheng Zhaob,QianXu
b, Qidong You a, b, Hua Xiang a, b,
*
aJiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University,
24 Tongjiaxiang, Nanjing 210009, PR China
bDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University,
24 Tongjiaxiang, Nanjing 210009, PR China
* Corresponding author. Tel.: +86 025 83271096; fax: +86 025 83271096.
E-mail address: xianghua@cpu.edu.cn (H. Xiang).
Abstract
Various 3-substituted 4-anilino-coumarin derivatives have been designed, synthesized
and their anti-proliferative properties have been studied. The in vitro cytotoxicity screening
was performed against MCF-7, HepG2, HCT116 and Panc-1 cancer cell lines by MTT assay.
Most of the synthesized compounds exhibited comparable anti-proliferative activity to the
positive control 5-Fluorouracil against these four tested cancer cell lines. Among the different
substituents at C-3 position of coumarin scaffold, 3-trifluoroacetyl group showed the most
promising results. Especially, compounds 33d (IC50=16.57, 5.45, 4.42 and 5.16 μM) and 33e
(IC50=20.14, 6.71, 4.62 and 5.62 μM) showed excellent anti-proliferative activities on MCF-7,
HepG2, HCT116 and Panc-1 cell lines respectively. In addition, cell cycle analysis and
apoptosis activation revealed that 33d induced G2/M phase arrest and apoptosis in MCF-7
cells in a dose-dependent manner. Low toxicity of compounds 33d and 33e was observed
against human umbilical vein endothelial cells (HUVECs), suggesting their acceptable safety
profiles in normal cells. Furthermore, the results of in silico ADME studies indicated that both
33d and 33e exhibited good pharmacokinetic properties.
Keywords: coumarin derivatives; synthesis; antitumor; apoptosis; selectivity index.
Cancer is a worldwide disease that affects around fourteen million people every year. The
American Cancer Society estimated that 1,685,210 new cancer cases and 595,690 cancer
deaths will occur in the United States in 2016.1 Most of cancers are known as uncontrolled
growth of abnormal cells due to the dysfunction of essential enzymes and other proteins
controlling cell division and proliferation.2, 3 Although several therapeutic regimes have been
successfully approached for fighting cancer nowadays, cancer is still the second leading cause
of deaths worldwide.4 Until now, chemotherapy remains the principal strategy for the
treatment of cancer diseases.5 Accordingly, the discovery of new anticancer agents with
promising bioactivity and high therapeutic index is still an urgent need.
Natural products and its metabolites are important sources of novel antitumor agents.
Finding lead compounds from natural products is an effective way to develop novel antitumor
agents. This is due to the natural products' intrinsic and well defined biological activity, which
can be potentiated or modulated by structural optimization.
Coumarins, the most important classes of benzopyrones, are a wide group of natural
compounds. They are thought to be a significant source of inspiration for new anticancer

agents.6 They exhibit a variety of biological activities including anticancer,7-9 anti-HIV,10
anti-microbial,11 anti-inflammatory activity12 and so on. The antitumor effects, which are
related to the inhibition of the cellular proliferation, through a variety of mechanisms, were
extensively examined.13-16 Due to their potential applications in cancer therapy, a lot of efforts
have been performed aiming at designing properly functionalized synthetic coumarin
derivatives with improved anticancer activity.17, 18 Among them, coupling coumarin with
different bioactive molecules is one of the effective ways. Adopting this approach, some
4-substituted coumarins have exhibited significant antitumor activities. For example, 4-aryl
coumarin derivative (1, Fig. 1), was reported to show potent inhibition of tubulin
polymerization activity and cytotoxicity against HBL100 Cells with IC50 value of 88 nM.19
Stilbene-coumarin hybrid (2, Fig. 1), initially identified by Federica Belluti as a proapoptotic
agent, exerted an anti-proliferative effect in H460 cells by inducing apoptosis.20
Triphenylethylene-coumarin hybrid (3, Fig. 1) containing two amino side chains showed a
broad-spectrum and excellent antitumor activity by acting on DNA.21 (1, 2,
3-triazol)-coumarin hybrid (4, Fig. 1) with IC50 value of 0.52 μM against A549 cells induces
G2/M-phase cell cycle arrest.22
Figure 1. Structures of known coumarin derivatives
Similarly, considerable attention has been also focused on aniline moieties which have
been widely used as functional groups in medicinal chemistry as exemplified in Figure 2.
4-anilino-2-phenylquinazoline (5, Fig. 2) was reported by Michael Wiese and coworkers as a
selective inhibitor of Breast Cancer Resistance Protein ABCG2.23 4-biarylamino-quinazoline
(6, Fig. 2) was found to inhibit kinase activities of EGFR, FGFR-1 and PDGFRβ at
submicromolar concentrations and also showed EC50 values against several cancer cell lines
in
nanomolar
range.24
Additionally,
Kuo-Hsiung
Lee
et
al.
reported
4-(3-methylphenylamino)-2H-benzo[h]chromen-2-one (7, Fig. 2) endowed with potent
cytotoxic effect with ED50 value of 0.14 μM against A549 cells, which was a
coumarin-containing compound.25 So far few 4-anilino-coumarins have been investigated as
apoptosis inducers. In 2016, Lijuan Chen and his coworkers reported compound 8 and its
analogues, which showed potent and broad-spectrum in vitro and in vivo cytotoxic activities.26
Therefore, more efforts are expected to discover novel 4-anilino-coumarin derivatives
possessing improved activities.

Figure 2. Structures of known compounds bearing aniline moieties
Inspired by the promising research results mentioned above, we set out to design and
synthesize a number of novel heterocyclic compounds bearing coumarin scaffold and aniline
moieties (Fig. 3). In order to extend investigation to the coumarin derivatives, aromatic groups
such as 4-methoxyphenyl and aliphatic groups such as trifluoroacetyl were introduced to the
C-3 position of coumarin nucleus (Fig. 3). Furthermore, the effect of different substituent
groups (H, OMe or OH) at ring A on proliferative activity was also evaluated. Herein, we
described in this paper the synthesis of twenty-seven compounds of 4-anilino-coumarins with
3-(4-methoxyphenyl) group and nine compounds with 3-trifluoroacetylgroup. Cytotoxic
activity in vitro was examined against MCF-7 (breast carcinoma), HepG2 (liver carcinoma),
HCT116 (colon cancer), Panc-1 (pancreatic carcinoma) and HUVEC (human umbilical vein
endothelial cells). The effect of the most active compound 33d on triggering cell death events
was also assessed by flow cytometry assays. In silico ADME analysis was also performed to
predict important pharmacokinetic parameters of 33d and 33e.
Figure 3. Structures of proposed 3-substitued 4-anilino-coumarin derivatives
The synthetic routes of compounds 14(a-i) were shown in Scheme 1. Intermediate 11
was easily obtained from methyl salicylate 9 and 4-methoxyphenylacetic acid 10 through the
synthetic strategy depicted in literature.27 The 3-(4-methoxyphenyl)-4-hydroxy coumarin 12
was prepared from intermediate 11 via intramolecular Claisen condensation.28 Further
treatments with TsCl in the presence of TEA afforded the 3-(4-methoxyphenyl)-4-tosyloxy
coumarin 13. The target compounds 14(a-i) were generated by nucleophilic substitution of 13
with nine kinds of anilines.

Scheme 1. Reagents and conditions: (a) POCl3, pyridine, 0-10 °C, 3 h; (b) KOH, pyridine, rt, 6 h; (c)
TsCl, Et3N, dry DCM, rt, 0.5 h; (d) aromatic amines, K2CO3, EtOH, 75 oC, 2 h.
The synthesis of the target compounds 23(a-i) and compounds 25(a-i) were achieved by
employing the corresponding 4-methoxy deoxybenzoin 17 and 4-benzyloxy deoxybenzoin 18
as key intermediates (Scheme 2) which were prepared from commercially available
resorcinol and phenylacetic acids as starting materials. Briefly, resorcinol reacted with
4-hydroxybenzoic acid to yield benzophenone 16 under Fries reaction conditions. Compound
16 was then converted to the corresponding 4-substituted deoxybenzoin 17 or 18 through
selective alkylation of one hydroxyl with methanol or benzyl alcohol via Mitsunobu reaction
in 88% and 85% yield respectively. Further treatment of 17 and 18 with diethyl carbonate
(DEC)/NaH in reflux toluene gave compound 19 and 20, which were then converted to the
corresponding compounds 21 and 22 by reacting with TsCl and TEA in DCM. Similarly, the
target compounds 23(a-i) and intermediates 24(a-i) were prepared by nucleophilic
substitution with nine kinds of anilines. Finally, the final compounds 25(a-i) were achieved
via Bn-deprotection with Pd-C in THF under the condition of H2.

Scheme 2. Reagents and conditions: (a) BF3-OEt2, 80oC, 3 h; (b) MeOH for 17 or BnOH for 18, PPh3,
DIAD, THF, 0 oC-rt, 0.5 h; (c) NaH, DEC, 120 oC, 3 h; (d) TsCl, Et3N, dry DCM, 0.5 h; (e) aromatic
amines, K2CO3, EtOH, 75 oC, 2 h; (f) Pd-C, H2, THF, rt, 12 h.
The derivatives 32(a-d) and 33(a-e) were synthesized following the general procedures
as detailed in Scheme 3. Compounds 28 and 29 were respectively furnished by the cyclization
of 26 and 27 with diethyl carbonate (DEC) and NaH in reflux toluene. According to the
29
reported procedure, the following compound 30 and 31 were generated by a one-pot
procedure containing three steps. The final compounds 32(a-d) and 33(a-e) were synthesized
by the nucleophilic substitution with corresponding aniline groups in DMF.
Scheme 3. Reagents and conditions: (a) NaH, DEC, 120 oC, 3 h; (b) TMSiCl, dry pyridine, dry
1,4-dioxane, rt, 1 h; (c) (CF3CO)2O, 80-90oC, 2 h; (d) POCl3, 60oC, 2 h; (e) aromatic amines, K2CO3,
DMF, 75 oC, 2 h.
In vitro cytotoxic activity of all the synthesized 3-substituted 4-anilino-coumarin
derivatives 14(a-i), 23(a-i), 25(a-i), 32(a-d) and 33(a-e) in this paper was evaluated against
four human cancer cell lines (MCF-7, HepG2, HCT116 and Panc-1) using 5-Fluorouracil
(5-Fu) as the reference drug.30 The biological results of these synthesized compounds were
summarized in Table 1. From the results, it is evident that most of the tested compounds

displayed moderate anti-proliferation activities with IC50 values less than 100 μM. Among
them, fourteen compounds exhibited similar or better bioactivities in vitro compared to 5-Fu.
On the other hand, compounds 14(a-i), 23(a-i) and 25(a-i) with 4-methoxylphenyl at C-3 and
hydroxyl at C-7 position were found to be inactive except 25f, which suggested that hydroxyl
group may not be favored at C-7 position. Among those compounds with R1 substituted by H
or OMe, compounds 14e and 23e, comprising of hydroxyl at meta-position and methyl at
para-position of substituted anilines, displayed significant cytotoxicity with IC50 value
ranging from 9.75 to 12.34 μM and from 22.52 to 34.39 μM respectively against MCF-7,
HepG2, HCT-116 and Panc-1 cell lines. An enhanced effect was observed by the replacement
of 4-methoxyphenyl with trifluoroacetyl at C-3 position of coumarin (compare 23c and 23d
with 32c and 33d). The compounds bearing trifluoroacetyl at C-3 position such as compounds
(32b, 32c, 32d, 33b, 33d and 33e), showed significant anti-cancer activities with IC50 value
of 9.74 μM for compound 32c against MCF-7, 5.45 μM for compound 33d against HepG2,
4.42 μM for compound 33d against HCT116 and 5.16 μM for compound 33d against Panc-1
respectively. The analysis of the structures of analogues 32(a-d) and 33(a-i) revealed that the
intramolecular hydrogen bond between NH group and C=O group plays an important role in
cytotoxic activity. According to previous reports,31-33 the “endo enamine-ketone” conjugated
coumarin ring might serve as a bioisosteric replacement for the olefin functionality of the
cis-stilbene combretastatin structure. Due to the structural similarity with combretastatin
analogues, the promising antiproliferative activities of 33d and 33e might be achieved through
tubulin inhibition.
Table 1
In vitro cytotoxic activities of synthesized compounds 14(a-i), 23(a-i), 25(a-i), 32(a-d) and 33(a-e)
Cell lines (IC50, μM) a, b
Compound R1
R2
R
MCF-7
34.58
82.34
38.45
>100
HepG2
35.19
77.26
35.52
>100
10.16
31.32
59.41
>100
66.43
39.56
84.26
>100
HCT116 Panc-1
H
-
-
-
-
-
-
-
-
-
-
-
-
4-OMe
3, 4, 5-OMe
4-F
36.61
63.54
39.44
N.T. c
12.34
29.49
64.79
N.T.
37.45
77.59
32.02
N.T.
14a
14b
14c
14d
14e
14f
H
H
H
-
H
3-OH, 4-Me
4-OEt
12.08
30.66
65.31
>100
9.75
H
32.85
66.07
N.T.
H
4-Me
14g
14h
14i
H
4-NHCOCH3
3-CF3
H
21.62
39.25
89.45
>100
55.73
35.15
N.T.
49.08
41.02
N.T.
OCH3
OCH3
OCH3
4-OMe
3, 4, 5-OMe
4-F
23a
23b
23c
N.T.
N.T.

OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
-
-
>100
34.39
24.31
66.88
85.75
36.22
>100
55.98
71.46
89.55
>100
32.46
85.47
>100
49.09
55.73
19.21
9.74
>100
22.52
31.22
74.36
>100
57.25
78.76
49.65
65.63
79.02
>75.32
36.52
88.34
>100
53.56
38.38
26.43
8.26
N.T.
N.T.
23d
23e
23f
-
3-OH, 4-Me
4-OEt
26.48
33.03
82.44
N.T.
31.29
36.07
76.53
N.T.
-
-
4-Me
23g
23h
23i
-
4-NHCOCH3
3-CF3
-
41.16
N.T.
39.52
N.T.
-
4-OMe
3, 4, 5-OMe
4-F
25a
25b
25c
25d
25e
25f
OH
-
64.24
66.12
41.21
N.T.
63.91
75.05
48.25
N.T.
OH
-
OH
-
-
OH
-
3-OH, 4-Me
4-OEt
OH
-
46.10
N.T.
43.62
N.T.
OH
-
4-Me
25g
25h
25i
OH
-
4-NHCOCH3
3-CF3
N.T.
N.T.
OH
-
55.88
61.24
16.98
11.74
12.49
N.T.
67.21
47.49
24.11
12.23
15.76
N.T.
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
4-OMe
3, 4, 5-OMe
4-F
32a
32b
32c
32d
33a
33b
33c
33d
33e
5-Fu
-
37.40
>100
15.04
>100
16.57
20.14
26.14
10.45
>100
12.28
>100
5.45
OCH3 4-OMe
OCH3 3, 4, 5-OMe
OCH3 4-F
H
19.46
N.T.
23.83
N.T.
H
H
OCH3
-
4.42
5.16
H
OCH3 3-OH, 4-Me
6.71
4.62
5.62
-
-
-
49.02
38.30
47.83
a
Cytotoxicity as IC50 values for each cell line, the concentration of compound that inhibit 50% of the
cell growth after 48 h of drug exposure measured by MTT assay.
b Each value was reproduced in triplicate.
c Not test.
The results indicated that the designed 3-substitued 4-anilino-coumarin derivatives
exhibited potent anticancer activities. Based on the preliminary cytotoxicity results, we could
discuss a clear tendency of structure–activity relationship: (i) for C-3 position substituted
coumarin derivatives, placement of a trifluoroacetyl in place of the 4-methoxyphenyl
remarkably enhanced the cytotoxic potential; (ii) the coumarin analogs bearing trifluoroacetyl
at C-3 position and methoxy at C-7 position such as 32c with a 4-fluoroanilino group showed
much higher activity than the 4-methoxyanilino, 3, 4, 5-trimethoxyanilino and 3,
4-methylenedioxyanilino derivatives (32a, 32b and 32d); (iii) concerning analogs bearing
trifluoroacetyl at C-3 position and methoxy at C-6 position of coumarin, 33d with a 3,
4-methylenedioxyanilino group showed much higher activity than the 4-methoxyanilino, 3, 4,
5-trimethoxyanilino, 4-fluoroanilino and 3-hydroxy-4-methylanilino derivatives (33a, 33b,
33c and 33e). Especially, among all the designed compounds, 33d was found to be the most
potent compound against 4 human tumor cell lines. The structure–activity relationship (SAR)
results were summarized in Scheme 4.

Scheme 4. Structure–activity relationship of designed compounds
Many cytotoxic compounds exerted their anti-proliferative effect by either arresting the
cell cycle at a particular checkpoint of cell cycle or induction of apoptosis, or by a combined
effect.34 Regulation of the cell cycle and apoptosis are considered to be effective cancer
therapeutic methods. Now that compound 33d showed the highest anti-proliferative activity
among all the synthesized compounds, it was selected to assess the effect on cell-cycle arrest
of MCF-7 by flow cytometry assay using propidium iodide (PI) staining. As is seen from
Figure 4, compound 33d concentration-dependently arrested cell cycle at G2/M phase,
accompanied with decrease of cells at G0/G1 phase. Specifically, the percentage of cells at
G2/M phase for the high concentration group (15 μM) was 63.06±2.24%, about 39% higher
than that of the control group (***P < 0.001).
In order to identify whether 33d induced cell death through apoptosis, a morphological
observation study was used to evaluate its influence on cell skeleton and Hoechst 33258
staining was taken to evaluate nuclei condensation. Under the inverted light microscope
(x200), incubation of 5 μM, 10 μM and 15 μM of 33d for 24 h resulted in phenotypic changes
of MCF-7 cells, such as distortion, membrane blebbing, shrinkage and rounding up (arrow
pointing), while cells in untreated group grew well (Figure 5).These results were also
confirmed on MCF-7 cells using Annexin V-PI staining by flow cytometry. It was observed
that 33d induced apoptosis against MCF-7 cells in a dose-dependent manner as shown in
Figure 6. Results indicated that there was increased apoptotic activity in MCF-7 cells treated
with 33d at increasing concentrations (5, 10 and 15 μM) for 48 h. Specifically, when treated
with 33d at high concentration (15 μM), around 32.1% of early apoptosis rate were observed.

Figure 4. (A) Cell cycle analysis of MCF-7 cells treatment of compound 33d (5, 10, 15 μM) and no
treatment (DMSO) as reference control for 48 h. (B) Histogram provides percentage of cells in G0/G1
and G2/M phases of the cycle calculated by flow cytometer. Results are expressed as mean ± SEM of
three independent experiments. ***P < 0.001 indicates significant statistical difference from untreated
control cells.
Figure 5. Morphological changes (A) and nuclear degradation (B) of MCF-7 cells induced by 33d. The
arrow pointed to apoptotic bodies.
Figure 6. Flow cytometric apoptosis staining analysis of MCF-7 cells after 48 h treatment with 33d (5,
10, 15 μM) and no treatment (DMSO): viable (lower left), early apoptotic (lower right), late apoptotic
(upper right), and necrotic cells (upper left).

One of the major hindrances for druggability of compounds with effective antitumor
activity is their toxicity to normal cells.35 Thus, it is necessary to evaluate cytotoxicity on
normal cells in the anticancer drug study. The promising compounds 33d and 33e were
chosen for selectivity test on normal human umbilical vein endothelial cells (HUVEC) using
the MTT assay. The selectivity indexes (SI) were calculated through IC50 values in cancer
cells divided by IC50 values in normal cells. The results revealed that the two tested
compounds were less sensitive to normal cells over cancer cells with SI > 4 (Table 2). The
low toxicity and selectivity warrant their further development for identifying more potent
anticancer agents.
Table 2
In vitro antiproliferative activities of compounds 33d and 33e against normal cell line (HUVEC)
Cell line (IC50, μM) a, b
Selectivity index c
Compound
HUVEC
26.27
MCF-7
1.59
HepG2
HCT116
5.94
Panc-1
5.09
4.82
4.39
33d
33e
29.43
1.46
6.37
5.23
a Antiproliferative effect (IC50) of HUVEC cell line, the concentration of compound that inhibit 50% of
the cell growth after 48 h of drug exposure measured by MTT assay.
b Each value was reproduced in triplicate.
c Selectivity index representing IC50 for normal cell line/IC50 for cancerous cell lines.
Employing Schrödinger software, compounds 33d and 33e were subjected to the
analyses of several physiochemical properties (ADME) related to pharmacokinetics36. Both
the compounds follow Lipinski’s rule of five for good bioavailability. With the
calculated
LogP values less than 5, these two compounds have good hydrophilicity with moderate
lipophilicity and hence should be able to gain access to membrane surfaces. The aqueous
solubility (LogS) of a compound is a major driving force that leads to good absorption and
distribution characteristics. The calculated LogS values of 33d and 33e were within the
acceptable range. Other calculations related to solubility, serum protein binding, blood–brain
barrier (LogBB and apparent MDCK cell permeability), gut–blood barrier (Caco-2 cell
permeability), number of likely metabolic reactions, skin permeability (Kp), and human oral
absorption in the gastrointestinal tract showed these values for both 33d and 33e within the
standard ranges for good bioavailable drugs (Table 3).
Table 3
Various physicochemical (ADME) parameters calculated for 33d and 33e
Compound
Parameter
Stand. range*
33d
33e
5-Fu
LogS for aqueous solubility
LogP
-2.832 -4.244 -1.073
2.563 3.056 -0.885
-0.402 0.115 -0.739
-0.191 -0.642 -0.955
(-6.5/0.5)
(-2.0/6.5)
(-1.5/1.5)
(-3.0/1.2)
(1.0/8.0)
LogK hsa for serum protein binding
LogBB for brain/ blood
No. of metabolic reactions
3
4
0

Apparent Caco-2 permeability (nm/s)
Apparent MDCK permeability (nm/s)
LogKp for skin permeability
1584
2314
691
937
120
87
(<25 Poor, >500 great)
(<25 Poor, >500 great)
(-8.0 to -1.0, Kp in cm/h)
(<25% is poor)
-1.976 -2.629 -5.053
100 96 59
% Human oral absorption in GI (±20%)
Qual. Model for human oral absorption
HIGH HIGH Medium (>80% is high)
* Note: for 95% of known drugs based on Schrödinger, QikProp v4.4 (2015) software results.
In summary, a series of 3-substitued 4-anilino-coumarin derivatives were synthesized,
characterized and evaluated for their in vitro cytotoxic effect on MCF-7, HepG2, HCT116 and
Panc-1 cell lines. Some compounds exhibited better antiproliferative activities against the
tested cells than positive control (5-Fluorouracil). Among them, compound 33d bearing
trifluoroacetyl at C-3 position and 3, 4-methylenedioxyphenylamino at C-4 position of
coumarin scaffold showed broad-spectrum and excellent anti-proliferative activities with IC50
in low 4 μM range. The further mechanism study demonstrated that compound 33d could
obviously induce cell cycle arrest and apoptosis in MCF-7 breast carcinoma cells. Moreover,
compounds 33d and 33e exhibited low toxicity against HUVECs, suggesting their acceptable
safety profiles in normal cells. In silico studies showed that both 33d and 33e possessed good
physiochemical properties (ADME) related to pharmacokinetics. Therefore, these two
compounds could serve as promising lead candidates for further study.
Acknowledgments
Research reported in this publication was supported by the NSFC (81373279), the
Twelfth Five-Year Plan Major Project of Candidate Drugs (Ministry of National Science and
Technology, 2012ZX09103101048), the Jiangsu Province Science and Technology Support
Program of Social Development Projects (BE2012745) and the Innovation Project of Jiangsu
Province.
Supplementary data
Supplementary data (experimental procedures and characterization data) associated with
this article can be found, in the online version, at xxx.
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Graphical abstract