Structure-activity relationship studies and in vitro and in vivo anticancer activity of novel 3-aroyl-1,4-diarylpyrroles against solid tumors and hematological malignancies

Article abstract of DOI:10.1016/j.ejmech.2019.111828

Novel 3-aroyl-1,4-diarylpyrrole derivatives were synthesized to explore structure-activity relationships at the phenyls at positions 1 and 4 of the pyrrole. The presence of amino phenyl rings at positions 1 and 4 of the pyrrole ring were found to be a crucial requirement for potent antitumor activity. Several compounds strongly inhibited tubulin assembly through binding to the colchicine site. Compounds 42, 44, 48, 62 and 69 showed antitumor activity with low nanomolar IC₅₀ values in several cancer cell lines. Compound 48 was generally more effective as an inhibitor of glioblastoma, colorectal and urinary bladder cancer cell lines; 69 consistently inhibited CML cell lines and demonstrated superiority in nilotinib and imatinib resistant LAMA84-R and KBM5-T315I cells. In animal models, compound 48 exhibited significant inhibition of the growth of T24 bladder carcinoma and ES-2 ovarian clear cell carcinoma tumors. Compounds 48 and 69 represent robust lead compounds for the design of new broad-spectrum anticancer agents active in different types of solid and hematological tumors.

Full text of DOI:10.1016/j.ejmech.2019.111828

Journal Pre-proof
Structure-activity relationship studies and in vitro and in vivo anticancer activity of
novel 3-aroyl-1,4-diarylpyrroles against solid tumors and hematological malignancies
Michela Puxeddu, Hongliang Shen, Ruoli Bai, Antonio Coluccia, Marianna Nalli,
Carmela Mazzoccoli, Eleonora Da Pozzo, Chiara Cavallini, Claudia Martini, Viviana
Orlando, Stefano Biagioni, Cristina Mazzoni, Addolorata Maria Luce Coluccia, Ernest
Hamel, Te Liu, Romano Silvestri, Giuseppe La Regina
PII:
S0223-5234(19)30980-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.111828
EJMECH 111828
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 August 2019
Revised Date: 24 October 2019
Accepted Date: 26 October 2019
Please cite this article as: M. Puxeddu, H. Shen, R. Bai, A. Coluccia, M. Nalli, C. Mazzoccoli, E. Da
Pozzo, C. Cavallini, C. Martini, V. Orlando, S. Biagioni, C. Mazzoni, A.M.L. Coluccia, E. Hamel, T. Liu,
R. Silvestri, G. La Regina, Structure-activity relationship studies and in vitro and in vivo anticancer
activity of novel 3-aroyl-1,4-diarylpyrroles against solid tumors and hematological malignancies,
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111828.
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2019 Published by Elsevier Masson SAS.

Structure-Activity Relationship Studies and in Vitro and in Vivo Anticancer
Activity of Novel 3 Aroyl-1,4-diarylpyrroles against Solid Tumors and
Hematological Malignancies


a,#
b,#
Hongliang Shen,
c a a
Ruoli Bai, Antonio Coluccia, Marianna Nalli,
Michela Puxeddu
d
e
e
e
Carmela Mazzoccoli, Eleonora Da Pozzo, Chiara Cavallini, Claudia Martini, Viviana
fi
f
f
g
Orlando, Stefano Biagioni, Cristina Mazzoni, Addolorata Maria Luce Coluccia, Ernest
c
h,* a,* a,*
Hamel, Te Liu,
Romano Silvestri, and Giuseppe La Regina,
a
Laboratory affiliated with the Institute Pasteur Italy – Cenci Bolognetti Foundation,
Department of Drug Chemistry and Technologies, Sapienza University of Rome, Piazzale Aldo
Moro 5, I-00185 Roma, Italy
b
Department of Urology, Capital Medical University Beijing Friendship Hospital, Beijing,
China, 100050
c
Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer
Treatment and Diagnosis, Frederick National Laboratory for Cancer Research, National Cancer
Institute, National Institutes of Health, Frederick, Maryland 21702, United States
d
Laboratorio di Ricerca Pre-Clinica e Traslazionale, Istituto di Ricovero e Cura a Carattere
Scientifico (IRCCS), Centro di Riferimento Oncologico della Basilicata, Via Padre Pio 1, I-
8
5028 Rionero in Vulture, Italy
e
Department of Pharmacy, University of Pisa, Via Bonanno Pisano 6, I-56126 Pisa, Italy
f
Department of Biology and Biotechnologies “Charles Darwin”, Sapienza University of Rome,
Piazzale Aldo Moro 5, I-00185 Roma, Italy
g
Department of Biological and Environmental Sciences and Technologies, University of
Salento, I-73100 Lecce, Italy
h
Shanghai Geriatric Institute of Chinese Medicine, Shanghai University of Traditional Chinese
Medicine, 365 South Xiangyang Road, Shanghai, China, 200031

Keywords:
Tubulin
Leukemia
Solid tumor
Inhibitor
Pyrrole
#
First co-authors
M.P. e-mail address: michela.puxeddu@uniroma1.it; phone: +39 06 4991 3404
H. S. e-mail address: 1051486940@qq.com; phone: +86 1391 1859 490
*
Corresponding Authors
G.L.R. e-mail address: giuseppe.laregina@uniroma1.it; phone: +39 06 4991 3893
R.S. e-mail address: romano.silvestri@uniroma1.it; phone: +39 06 4991 3800
T.L. e-mail address: 0721160004@mail.tongji.edu.cn; phone: +86 21 64720010

ABSTRACT
Novel 3-aroyl-1,4-diarylpyrrole derivatives were synthesized to explore structure-activity
relationships at the phenyls at positions 1 and 4 of the pyrrole. The presence of amino phenyl
rings at positions 1 and 4 of the pyrrole ring were found to be a crucial requirement for potent
antitumor activity. Several compounds strongly inhibited tubulin assembly through binding to
the colchicine site. Compounds 42, 44, 48, 62 and 69 showed antitumor activity with low
nanomolar IC50 values in several cancer cell lines. Compound 48 was generally more effective as
an inhibitor of glioblastoma, colorectal and urinary bladder cancer cell lines; 69 consistently
inhibited CML cell lines and demonstrated superiority in nilotinib and imatinib resistant
LAMA84-R and KBM5-T315I cells. In animal models, compound 48 exhibited significant
inhibition of the growth of T24 bladder carcinoma and ES-2 ovarian clear cell carcinoma tumors.
Compounds 48 and 69 represent robust lead compounds for the design of new broad-spectrum
anticancer agents active in different types of solid and hematological tumors.

1
. Introduction
Cancer is a serious leading cause of death worldwide, claiming more than 8.2 million human
lives in 2012. According to available global cancer statistics, lung (12.3%), breast (12.3%),
colorectal (10.6%) and prostate (7.5%) account for the most cancers diagnosed worldwide.
Nevertheless, leukemia alone represents 2.5 % of all diagnosed cancers [1,2]. Cancer cells are
characterized by a high rate of cell division. Microtubules (MTs) play a key role in regulating the
3
eukaryotic cell machinery. Interfering with the MT dynamic equilibrium is an attractive option
for the design of effective agents against a wide variety of cancers [4-11].
We recently reported the synthesis of (4-(4-aminophenyl)-1-phenyl-1H-pyrrol-3-yl)(3,4,5-
trimethoxyphenyl)methanone (1) (Chart 1), an inhibitor of tubulin polymerization and MCF-7
cancer cell growth with antiproliferation activity in BCR/ABL-expressing chronic myeloid
leukemia (CML) cells from patients in blast crisis [12]. Herein we report the synthesis and
structure-activity relationship (SAR) studies of new 3-aroyl-1,4-diarylpyrrole (ARDAP)
compounds 2-69. The SAR studies have been grouped in three series of derivatives based on the
substituents (i) at the pyrrole 1-phenyl ring (Ring A, 2-16), (ii) at the 4-phenyl ring (Ring B, 17-
3
3), and (iii) at both these phenyl rings (Rings A+B, 34-69). We examined their effects as
inhibitors of tubulin polymerization and MCF-7 breast cancer cell growth and as inhibitors of the
binding of colchicine to tubulin (Tables 1-3). Five highly potent derivatives, 42, 44, 48, 62 and
6
9, were evaluated in a panel of cancer cell lines, including leukemia (KU812, LAMA84-S,
LAMA84-R, KBM5-WT, KBM5-T315I), glioblastoma (T98G, U87MG and U343MG),
neuroblastoma (SK-N-BE and SK-N-BE(2)-C), colorectal (HT29, HCT116, SW480 and SW620)
and urinary bladder (T24) cells. ARDAPs 42, 44, 48, 62 and 69 demonstrated broad spectrum
activity in all these cell lines, with 48 generally more effective in the glioblastoma, colorectal

and urinary bladder cancer cells and 69 in the leukemia cell lines. In animal models, compound
8 exhibited significant inhibition of the growth of the T24 bladder and ES-2 ovarian clear cell
carcinoma tumors.
4
H N
2
OMe
OMe
OMe
OMe
OMe
OMe
O
O
N
N 1
A
R
2
-16 (Ring A)
1
OMe
OMe
OMe
OMe
OMe
OMe
R
R2
B
O
B
O
4
4
N
N1
A
R1
1
7-33 (Ring B)
34-69 (Ring A+B)
Chart 1. General structures of ARDAP compounds 1, 2-16 (Table 1), 17-33 (Table 2 and 34-69 (Table 3).
2
. Chemistry
2
.1. Synthetic procedures
The new ARDAP derivatives were synthesized as depicted in Scheme 1. Reaction of an
appropriate benzaldehyde with 3,4,5-trimethoxyacetophenone (70) in the presence of NaOH in
ethanol at room temperature for 24 or 2 h afforded chalcones 71-84 and 85-89, respectively,
which underwent cyclization into the corresponding pyrrole derivatives 90-108 with p-
toluenesulfonylmethyl isocyanide (TosMIC) in the presence of NaH in dimethyl sulfoxide and
diethyl ether at room temperature. In order to improve yield, pyrroles 90-108 were transformed

into ARDAP compounds by different procedures: (i) with an appropriate boronic acid in the
presence of copper(II) acetate and triethylamine in dichloromethane under an Ar stream at room
temperature (2, 26, 30 and 33), at 40 °C (3, 7, 24, 58) or at 50 °C (17-23, 25, 27-29, 31, 32, 34-
3
8, 41, 49-53, 56, 57, 59 and 60) for 18 h, or in 1,2-dichloroethane at 80 °C for 24 h (8, 10-12
and 15); for compound 6, pyridine in dichloromethane was used instead of triethylamine at room
temperature for 5 h; (ii) with an appropriate iodonitrobenzene, copper(I) bromide, cesium
carbonate and 8-quinolinol N-oxide in dimethyl sulfoxide at 65 °C for 18 h (4, 9 and 13); (iii)
with an appropriate iodoaniline, copper(I) iodide, cesium carbonate, 1,10-phenanthroline in 1,4-
dioxane at 110 °C for 24 h (42-46 and 63-67); for 16, DMF at 110 °C overnight was used.
Reduction of nitro derivatives 4, 13, 37, 38, 45, 46, 53, 59, 60, 66, 67 with tin(II) chloride
dehydrate in ethyl acetate at 80 °C for 3 h furnished amino ARDAPs 5, 14, 39, 4, 47, 48, 54, 55,
6
1, 62, 68 and 69.
.2 Molecular modeling studies
2
The binding modes of compounds 42, 44, 48, 62 and 69 were evaluated by docking experiments
using our previously reported procedure [12]. The proposed docking poses were consistent
across all inspected structures and were superimposable with the bound DAMA-colchicine (pdb
code: 1SA0) [13]. Inspection of the binding mode highlighted the following key contacts: (i) the
trimethoyphenyl ring formed polar contacts with the Cys241β side chain and hydrophobic
interactions with Leu248β and Leu255β; (ii) the B ring was involved in hydrophobic contacts
with the Lys352β side chain and π-cation interactions with the Lys352 ε-nitrogen atom; (iii) the
A ring formed hydrophobic interactions with Met259β, Lys353β, Ala 180α and Val181α. The
position of the substituent of both the A and B rings did not affect the binding mode (Figure
1
SD, supplementary data).

O
O
O
OMe
OMe
OMe
OMe
Me
a
H
R2
R2
+
OMe
OMe
7
0
71-89
HO
B
R2
OMe
R2
OMe
OH
O
O
OMe
OMe
OMe
R1
b
OMe
N
N
c
H
R1
9
0-108
I
I
2
4
,
1
3
,
,
6-8
,
10-12
,
15
,
17-38,
46
,
49-53, 56-60
d
NO2
OMe
NH2
e
R2
OMe
O
R2
O
OMe
OMe
OMe
OMe
N
N
R1
R1
1
6, 42-46, 63-67
4
,
9
,
13
R2
OMe
OMe
OMe
O
f
4
5
, 13
,
37, 38
,
45, 46
, 53,
9
,
60, 66
,
67
(
R , R = NO )
1 2 2
N
R1
, 14, 39, 40, 47, 48, 54, 55, 61, 62, 68, 69
5
Scheme 1. Synthesis of compounds 2-69. For structures 2-69, see Tables 1-3. Compounds 71-108, R : H,
2
7
1, 90; 2-Me, 85, 91; 2-Cl, 72, 92; 3-Cl, 86, 93; 4-Cl, 73, 94; 2-Br, 74, 95; 3-Br, 75, 96; 4-Br, 76, 97; 2-
NO , 77, 98; 3-NO , 78, 99; 4-NO , 79, 100; 2-MeO, 87, 101; 3-MeO, 88, 102; 4-MeO, 80, 103; 5-Br,2-
2
2
2
MeO, 81, 104; 2,5-F
2 3
, 82, 105; 2,4,6-F , 89, 106; styryl, 83, 107; 2-F, 84, 108. Reagents and reaction
conditions: (a) appropriate benzaldehyde, NaOH, EtOH 96%; r.t., 24 h (71-84) or 2 h (85-89); (b) NaH,
TosMIC, DMSO/Et O, r.t., 4 h (90-108); (c) appropriate boronic acid, Cu(OAc) , Et N, DCM, r.t., 18 h
2
2
3
(
2, 26, 30, 33), DCM, 40 °C, 18 h (3, 7, 24, 58), DCE, 50 °C, 18 h (17-23, 25, 27-29, 31, 32, 34-38, 41,
9-53, 56, 57, 59 and 60), DCE, 80 °C, 18 h, (8, 10-12, 15), Pyr, DCM, r.t., 5 h (6); (d) appropriate
iodonitrobenzene, CuBr, 8-quinolinol N-oxide, Cs CO , DMSO, 65 °C, 18 h (4, 9, 13); (e) appropriate
4
2
3
iodoaniline, CuI, Cs
2 3
CO , 1,10-phenanthroline, dioxane, 110 °C , 24 h (42-46, 63-67), 110 °C, DMF,
.
overnight (16); (f) SnCl 2H O, MeCOOEt, 80 °C, 3 h (5, 14, 39, 4, 47, 48, 54, 55, 61, 62, 68 and 69).
2
2

Table 1
Inhibition of tubulin polymerization, growth of MCF-7 human breast carcinoma cells and colchicine
a
binding to tubulin by compounds 2-16.
OMe
O
OMe
OMe
N
A
R
2
-16
Compd
Ph-R (A ring)
IC ± SD (µM)
IC50 ± SD (nM)
(% ± SD)
5
0
b
c
d
Tubulin
MCF-7
Inh. colch. bind.
e
e
2
3
4
5
6
7
8
9
>20
2200 ± 50
1800 ± 200
5000 ± 0
3800 ± 400
150 ± 20
150 ± 4
nd
>20
>20
>20
nd
nd
nd
1.3 ± 0.01
2.1 ± 0.2
1.9 ± 0.04
>20
22 ± 4
37 ± 4
23 ± 4
12 ± 2
25 ± 2
42 ± 2
78 ± 4
18 ± 3
78 ± 4
550 ± 70
500 ± 70
140 ± 40
20 ± 2
1
1
1
1
0
1
2
3
1.6 ± 0.04
1.7 ± 0.1
0.66 ± 0.1
3.3 ± 0.3
4000 ± 700

1
1
4
5
1.6 ± 0.1
35 ± 7
67 ± 1
27 ± 5
0.96 ± 0.1
120 ± 10
1
6
>20
>5000
6.5 ± 4
a
b
c
d
e
Experiments were performed in duplicate or triplicate.
Inhibition of tubulin polymerization. Tubulin was at 10
µ
M in the assembly assay.
Inhibition of growth of MCF-7 human breast carcinoma cells.
3
3
Inhibition of [ H]colchicine binding: tubulin, [ H]colchicine, inhibitor at 1:5:5 µM.
nd, not done.

Table 2
Inhibition of tubulin polymerization, growth of MCF-7 human breast carcinoma cells and colchicine
a
binding to tubulin by compounds 17-33.
OMe
OMe
OMe
R
B
O
N
1
7-33
Compd
Ph-R (A ring)
IC ± SD (µM)
IC50 ± SD (nM)
(% ± SD)
5
0
b
c
d
Tubulin
MCF-7
Inh. colch. bind.
1
1
1
2
2
2
2
2
2
2
7
8
9
0
1
2
3
4
5
6
0.79 ± 0.1
0.63 ± 0.07
2.3 ± 0.04
1.2 ± 0.08
0.67 ± 0.1
>20
190 ± 30
76 ± 8
18 ± 1
50 ± 2
540 ± 90
290 ± 10
190 ± 10
950 ± 70
340 ± 60
49 ± 10
50 ± 10
590 ± 20
7.5 ± 0.3
23 ± 2
39 ± 1
e
nd
0.77 ± 0.1
0.73 ± 0.1
0.94 ± 0.06
14 ± 4
61 ± 1
12 ± 1
nd
O N
2
f
>20

2
2
2
7
8
9
0.55 ± 0.08
0.45 ± 0.05
0.48 ± 0.04
190 ± 30
180 ± 30
70 ± 10
31 ± 2
27 ± 1
42 ± 0.2
3
3
3
3
0
1
2
3
>20
2200 ± 50
230 ± 40
180 ± 30
2200 ± 50
nd
24 ± 0.07
27 ± 1
nd
1.3 ± 0.08
0.51 ± 0.1
>20
a
b
c
d
e
Experiments were performed in duplicate or triplicate.
Inhibition of tubulin polymerization. Tubulin was at 10
µ
M in the assembly assay.
Inhibition of growth of MCF-7 human breast carcinoma cells.
3
3
Inhibition of [ H]colchicine binding: tubulin, [ H]colchicine, inhibitor at 1:5:5 µM.
nd, not done.

Table 3
Inhibition of tubulin polymerization, growth of MCF-7 human breast carcinoma cells and colchicine
a
binding to tubulin by compounds 34-69.
OMe
OMe
OMe
R2
B
O
N
A
R1
3
4-69
Compd
Ph-R (A ring)
Ph-R (B ring)
IC ± SD (µM) IC ± SD (nM)
(% ± SD)
1
2
50
50
b
c
d
Tubulin
MCF-7
Inh. colch. bind.
3
3
3
3
3
3
4
4
4
4
4
4
4
5
6
7
8
9
0
1
2
3
4
5
0.79 ± 0.04
0.71 ± 0.1
1.2 ± 0.06
>20
190 ± 20
420 ± 30
220 ± 50
880 ± 40
57 ± 4
39 ± 3
41 ± 9
4.2 ± 5
10 ± 0.8
64 ± 4
88 ± 0.4
41 ± 2
90 ± 1
79 ± 2
82 ± 0.2
32 ± 5
>20
780 ± 200
130 ± 30
18 ± 4
0.91 ± 0.1
0.33 ± 0.02
1.1 ± 0.1
0.50 ± 0.02
0.63 ± 0.1
0.71 ± 0.02
1.7 ± 0.07
190 ± 10
17 ± 5
40 ± 4
14 ± 2
510 ± 80

4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
0.78 ± 0.1
0.88 ± 0.1
0.47 ± 0.03
0.78 ± 0.06
0.48 ± 0.04
0.61 ± 0.004
0.71 ± 0.07
0.65 ± 0.1
0.42 ± 0.01
0.36 ± 0.06
0.66 ± 0.1
0.51 ± 0.02
0.68 ± 0.1
>20
110 ± 7
60 ± 7
41 ± 2
70 ± 3
14 ± 2
92 ± 0.07
80 ± 0.5
70 ± 3
44 ± 9
180 ± 30
65 ± 7
71 ± 1
210 ± 7
200 ± 40
53 ± 4
36 ± 5
34 ± 2
88 ± 0.3
95 ± 0.9
68 ± 4
6.0 ± 2
39 ± 1
83 ± 10
38 ± 4
65 ± 0.6
46 ± 0.04
11 ± 4
440 ± 60
240 ± 30
53 ± 4
>20
0
0.41 ± 0.02
0.50 ± 0.1
0.59 ± 0.1
0.71 ± 0.1
68 ± 2
12 ± 3
90 ± 0.1
79 ± 1
28 ± 4
83 ± 10
65 ± 0.6

6
6
6
6
6
5
6
7
8
9
0.52 ± 0.01
3.8 ± 0.4
53 ± 4
260 ± 60
260 ± 30
78 ± 4
69 ± 0.6
18 ± 2
1.8 ± 0.3
23 ± 5
0.87 ± 0.1
0.38 ± 0.04
59 ± 2
15 ± 2
87 ± 0.09
a
Experiments were performed in duplicate or triplicate.
Inhibition of tubulin polymerization. Tubulin was at 10
b
µM in the assembly assay.
c
Inhibition of growth of MCF-7 human breast carcinoma cells.
d
3
3
Inhibition of [ H]colchicine binding: tubulin, [ H]colchicine, inhibitor at 1:5:5 µM. With the inhibitor at
µM, 42 yielded 68 ± 0.4, 48 67 ± 2, 55 74 ± 2, and 62 61 ± 4 % inhibition.
1
e
nd, not done.
3
. Results and discussion
3
.1. Inhibition of tubulin polymerization and MCF+7 cancer cell growth
3
.1.1. Compounds 2-16, Ring A (Table 1). We first synthesized ARDAP derivatives 2-16 to
explore a variety of substituents at the 1-phenyl of the pyrrole nucleus (Table 1). As tubulin
polymerization inhibitors, ARDAPs 6, 8, 10, 11 and 14 showed IC50 values in the 1.0-2.0 µM
range, and two compounds, 12 and 15, were inhibitory at submicromolar concentrations. With
the exception of 6, introduction of substituent at position 2 of the 1-phenyl ring abolished the
ability of a compound to inhibit tubulin polymerization. Derivatives bearing a methoxy group at
position 2, 3 or 4 of the 1-phenyl ring were all potent inhibitors of tubulin assembly (compare 6
(
IC = 1.3 µM), 10 (IC = 1.6 µM) and 15 (IC = 0.96 µM). Among ARDAPs 2-16, compound,
50 50 50
1
2 (IC = 0.66 µM) was the most potent tubulin polymerization inhibitor. In general, ARDAPs
50

bearing a substituent at position 4 of the 1-phenyl ring were superior to the corresponding 3-
substituted counterparts (compare 11 with 7, 12 with 8, and 15 with 10) in both biochemical and
cellular assays. The 4-bromo- 12 (IC50 = 20 nM) and 4-amino- 14 (IC50 = 35 nM) phenyl
derivatives were the most potent inhibitors of the growth of human MCF-7 nonmetastatic breast
cancer epithelial cells among the ring A modified derivatives.
3
.1.2. Compounds 17-33, Ring B (Table 2). ARDAP derivatives 17-33 were synthesized to
evaluate substituent on the 4-phenyl ring of the pyrrole nucleus (Table 2). Ten ARDAP
derivatives, 17, 18, 21, 23-25, 27-29 and 32, inhibited tubulin polymerization with IC50 values at
submicromolar concentrations, and two compounds (20 and 31) had IC50 values in the 1.0-2.0
µM range. ARDAP derivatives bearing a chlorine or a bromine atom at position 2 or 4 of the 4-
phenyl ring were more potent than the corresponding 3-substituted counterparts [compare 18
(
(
(
IC = 0.63 µM) and 20 (IC = 1.2 µM) with 19 (IC = 2.3 µM); 21 (IC = 0.67 µM) and 23
50 50 50 50
IC = 0.77 µM) with 22 (IC >20 µM)]. In contrast, ARDAPs 24 (IC = 0.73 µM) and 25
5
0
50
50
IC50 = 0.94 µM), bearing a nitro group at position 2 or 3 of the 4-phenyl ring were much
superior to the corresponding 4-nitro derivative 26 (IC50 >20 µM). Introduction of a methoxy
group on any position of the 4-phenyl ring (ARDAPs 27-29) yielded potent inhibitors of tubulin
assembly, with IC ’s of 0.55, 0.45 and 0.48 µM, respectively. Fluorine atoms at positions 2,5-
5
0
(
31, IC = 1.3 µM) or 2,4,6 (32, IC = 0.51 µM) of the 4-phenyl ring also resulted in potent
50 50
tubulin polymerization inhibitors. Several derivatives inhibited MCF-7 cancer cell growth with
high nanomolar IC values; compounds 18, 24, 25 and 29 were the most potent inhibitors of the
5
0
MCF-7 cell growth in this group of derivatives, with IC values ranging from 49 (24) to 76 nM
5
0
(18).
3
.1.3. Compounds 34-69, Rings A+B (Table 3). We synthesized compounds 34-69 to combine

substituents at both the aryl rings at position 1 (Ring A) and position 4 (Ring B) of the pyrrole
nucleus (Table 3). New ARDAPs with substituents at position 2 of the 1-phenyl ring were
synthesized because of the weak activity displayed by derivatives 2-6 (Table 1). Many of these
derivatives (34, 35, 39, 40, 42-44, 46-58, 61-65, 68 and 69) inhibited tubulin polymerization with
submicromolar IC50 values, and four compounds (36, 41, 45 and 67) had IC50 values in the
1
.0−2.0 µM range.
3
1
.1.3.1. ARDAPs 34-48: R substituent at position 3 of the 1-phenyl ring. The 3-amino
derivatives at the 1-phenyl ring 42-48 were generally highly potent inhibitors in both the
biochemical and cellular assays, with the exception of 45, which has a nitro group at position 3
of the 4-phenyl ring. ARDAPs 42, 44 and 48 were the most potent MCF-7 cancer cell growth
inhibitors of this group, with IC values of 17, 14 and 14 nM, respectively. When the Ring A
5
0
substituent was a 3-methyl group (34-40) or a 3-fluorine atom (41), there was a significant
reduction in antiproliferative activity, with the exception of 40 (IC = 18 nM). The derivative
5
0
with a 2-nitro group on the 4-phenyl ring (44, IC = 14 nM) was a strong inhibitor of the MCF-7
5
0
cancer cell growth. The presence of an amino group on Ring B positively affected both the
inhibition of tubulin assembly and MCF-7 cancer cell growth, in particular when the amino
group was at position 4 of Ring B (compare 40, IC of 18 nM. and 48, IC of 14 nM).
5
0
50
3
.1.3.2. ARDAPs 49-69: R substituent at position 4 of the 1-phenyl ring. With three exceptions,
1
5
9, 60 and 66, these ARDAPs were potent inhibitors of tubulin polymerization, with IC values
5
0
at submicromolar concentrations. The most potent derivatives were characterized by the presence
of a 3- or 4-amino group on the 4-phenyl ring (54, tubulin assembly IC50 = 0.42 µM, MCF-7 IC50
=
53 nM; 55, tubulin assembly IC = 0.36 µM, MCF-7 IC = 6.0 nM; 61, tubulin IC = 0.41
50 50 50
µM, MCF-7 IC50 = 53 nM; 62, tubulin assembly IC50 = 0.50 µM, MCF-7 IC50 = 12 nM; 68,

tubulin IC = 0.87 µM, MCF-7 IC = 78 nM; 69, tubulin assembly IC = 0.38 µM, MCF-7 IC
50
5
0
50
50
=
15 nM). The derivatives 49-55, with a 4-methyl substituent on Ring A, were all potent
inhibitors of tubulin assembly, with the Ring B substituents 2-fluoro (49), 2-nitro (51), 3-amino
54) and 4-amino (55) also being highly effective inhibitors of MCF-7 cancer cell growth.
(
Among the 4-fluorophenyl derivatives 56-62, introduction of a nitro group (59 and 60) yielded
compounds with little ability to inhibit tubulin polymerization and reduced antiproliferative
activity. Among this group of compounds, the best antiproliferative activity was observed with
5
6
7
6, 58 and, especially, 62, with an IC of 12 nM against the MCF-7 cells. Generally, ARDAPs
50
3-69, bearing a 4-aminophenyl moiety, showed potent inhibition of tubulin assembly and MCF-
cancer cell growth, with the exception of nitro compounds 66 and 67. In this group,
exceptional antiproliferative and antitubulin activity was observed in compound 69, with para-
amino substituents in both Rings A and B. A SAR summary of tubulin polymerization inhibition
and inhibition of MCF-7 cancer cell growth of compounds 2-69 is shown in Chart 2.

Chart 2.
SAR Summary for tubulin polymerization inhibition (TPI) and MCF-7 cancer cell growth
inhibition (CGI) by compounds 2-69.
OMe
OMe
R
O
B
O
OMe
OMe
Ring B
TPI: good
OMe
OMe
4
CGI: all rings, weak
N 1
A
N
Ring A
R
2-subst-Ph: TPI and CGI bad
3
4
-subst-Ph: TPI good, CGI bad
-subst-Ph: TPI and CGI good
2
-16
1
7-33
OMe
O
R2
B
Rings A Ring B
OMe
4
3
3
-Me-Ph, all, except NO : TPI good, CGI weak
2
-NH -Ph, all:
TPI good, CGI good
TPI vey good, CGI good
all, expt NO2: TPI very good, CGI good
4-NH -Ph, all, expt NO : TPI and CGI excellent
OMe
N 1
2
4-Me-Ph, all:
-F-Ph,
4
A
R1
2
2
3
4-69
3
3
.2. Inhibition of the binding of [ H]colchicine to tubulin. Compounds 2-69 were also examined
3
for potential inhibition of the binding of [ H]colchicine to tubulin. ARDAP compounds 12, 40,
4
2-44, 48, 49, 54, 55, 62, 63 and 69 yielded >75% inhibition of the binding reaction. Is should be
noted that only one (12) among 2-16 with Ring B = phenyl (Table 1) and none of compounds 17-
3
4
3 with Ring A = phenyl (Table 2) showed binding inhibition >75%. The most potent derivatives
2, 44, 48, 55, 62 and 69, Rings A and B = aryl (Table 3), showed good correlation between
binding inhibition with a range of 87% (69) – 95% (55) and inhibition of the MCF-7 cancer cell
3
growth with IC50 ≤17 nM. Compounds with 80-90% inhibition of [ H]colchicine binding
inhibited the growth of the MCF-7 cancer cell line with IC values in the range of 12-53 nM;
5
0
3
except for 50, compounds with 70-80% inhibition of [ H]colchicine binding inhibited the growth
of the MCF-7 cancer cells with IC50’s in the range of 28-65 nM.

3
.3. Inhibition of leukemia KU812, LAMA84-S, LAMA84-R, KBM5-WT, and KBM5-T315I cells.
Chronic myeloid leukemia (CML) is a myeloproliferative disorder of hematopoietic
stem/progenitor cells [14]. CML arises from reciprocal chromosome translocation
t(9;22)(q34;q11) leading to the Philadelphia (Ph) chromosome encoding for the hybrid
phosphoprotein tyrosine kinase P210BCR-ABL that activates downstream signals leading to the
pathogenesis of CML [15,16]. CML predominantly affects adults, although it can also occur in
young people, accounting for 20% of adult leukemias. The NIH SEER Program has estimated
8
990 new cases and 1140 deaths in 2019 in the United States [17].
Imatinib mesylate (IM, STI-571, Gleevec) is a specific BCR-ABL and c-ABL tyrosine kinase
inhibitor (TKI), and it represents the standard treatment for CML. Treatment with the drug leads
to stable clinical responses and extended overall survival in the majority of patients. However,
patients treated with IM develop clinical resistance [18] to the drug due to point mutations in the
catalytic domain of the BCR/ABL oncoprotein [19] or to bcr/abl gene amplification [20]. Such
events usually result in CML patients having a fatal blast crisis. The BCR/ABL threonine for
isoleucine mutation at the “gatekeeper” position 315, T315I, causes drug resistance to IM and
other tyrosine kinase inhibitors (TKIs) [21,22]. New agents to overcome the IM drug resistance
in CML cells are needed. MTs have received attention as a target for the development of
alternative therapy for CML [10,11]. Recently, prototypic ARDAP [12] and MPT [23,24]
compounds have been reported as potential agents to treat CML. However, this field requires
further exploration.

Table 4
Growth inhibition of KU812, LAMA84-S, LAMA84-R, KBM5-WT and KBM5-T315I cell lines
by compounds 42, 44, 48, 62, 69 and reference nilotinib.
a
Compd
IC ± SD (nM)
50
KU812
LAMA84-S
LAMA84-R
KBM5-WT
KBM5-T315I
4
4
4
6
6
2
4
8
2
9
14 ± 4
16 ± 2
8 ± 2
15 ± 1
18 ± 4
10 ± 4
20 ± 2
8 ± 3
14 ± 2
20 ± 2
12 ± 4
22 ± 2
10 ± 2
126 ± 21
18 ± 4
15 ± 3
14 ± 3
25 ± 2
10 ± 3
12 ± 5
22 ± 2
21 ± 2
16 ± 3
26 ± 3
12 ± 2
2421 ± 2
18 ± 3
5 ± 2
b
NLT
10 ± 2
11 ± 2
a
Cytotoxic concentrations for the indicated cell lines. Experiments were performed in triplicate.
Incubation time was 48 h.
b
NLT: Nilotinib.
ARDAPs 42, 44, 48, 62 and 69 were compared with nilotinib (NLT) in the BCR/ABL-
expressing KU812 cells and in the LAMA84-S cell line sensitive to IM and its paired IM
resistant LAMA84-R. NLT is a second-generation TKI approved for the treatment of IM-
resistant CML, and NLT was superior to IM as a first-line treatment in newly diagnosed CML
[25,26]. Compounds 42, 44, 48, 62 and 69 inhibited the KU812, LAMA84-S and LAMA84R cell
lines at low nanomolar concentrations (Table 4). NLT was comparable to 42, 44, 48, 62 and 69
in the KU812 and LAMA84-S cells but showed weaker inhibition of the LAMA84-R cell line.
At such nanomolar concentrations, peripheral blood mononuclear cells (PBMCs) isolated from
healthy donors were minimally affected (data not shown). ARDAPs 42, 44, 48, 62 and 69 also
inhibited the IM-sensitive KBM5 and IM-resistant KBM5-T315I cells with similar IC50
inhibitory concentrations. Most importantly, the activity of these compounds did not appear to be
affected by the T315I mutation; in contrast, NLT, showed weak inhibition of this cell line. As an
inhibitor of the IM-resistant LAMA84-R and KBM5-T315I cell lines, 69 (IC of 10 and 12 nM,
5
0

respectively) was 12.6 and 201.7 times superior to NLT.
The ability of 69 to potentiate the cytotoxic effects of NLT was assessed in human KBM5
CML cells. KBM5-WT and KBM5-T315I cells were treated for 48 h with 10 nM NLT in the
presence or absence of 10 nM 69 and analyzed by the MTT assay. Apoptosis evaluation by flow
cytometry (annexin V-stained cells) showed that 69 potentiates NLT-mediated cell death in both
KBM5-WT and KBM5-T315I CML cells. The NLT+69 drug combination was clearly more
effective than NLT or 69 alone in increasing the percentage of apoptotic cells from near 50% to
9
0% (Fig. 1).
Fig. 1. ARDAP 69 potentiates NLT-mediated CML cell death, including those cells that express the
T315I mutation. Cells were treated for 48 h and then assessed by flow cytometric analysis. Experiments
were performed in triplicate.

3
.4. Inhibition of growth of glioblastoma T98G, U87MG and U343MG and neuroblastoma SK-
N-BE and SK-N-BE(2)-C cell lines. Glioblastoma is a highly malignant and rapidly growing type
of brain cancer. It is usually aggressive and a relatively common brain tumor in adults [27]. A
major difficulty in treating glioblastomas is caused by different cell types found in these tumors.
Standard therapy includes surgical resection, followed by postoperative radiation therapy and
adjuvant chemotherapy with the alkylating agent temozolomide [28]. Treatments of U343MG
(human glioblastoma-astrocytoma), U87MG (human glioblastoma-astrocytoma) and T98G
(human glioblastoma) cells with increasing concentrations of compounds 42, 44, 48, 62 and 69
produced a dose-dependent inhibition of cell viability (Fig.s 2SD-4SD, supplementary data). The
IC values obtained were in the nanomolar concentration range in each cell line, and ranged
5
0
from 12 (48) to 69 (42) nM in U343MG cells, from 30 (44) to 61 (42) nM in U87MG cells, and
from 22 (62) to 54 (42) nM in T98G cells.
Neuroblastoma is the most common extracranial solid tumor occurring in children. As
inhibitors of the SK-N-BE (human neuroblastoma) and SK-N-BE(2)-C (human neuroblastoma
chemio-resistant derived from SK-N-BE) cell lines, compounds 42, 44, 48, 62 and 69 were one
order of magnitude less effective than they had been in the glioblastoma cell lines. Compound 48
was the most active in both neuroblastoma cell lines, with EC values of 221 (SK-N-BE cells)
5
0
and 56 nM (SK-N-BE(2)-C cells) (Table 5).

Table 5
Growth inhibition of U343G, U87MG, T98G, SK-N-BE and SK-N-BE(2)-C cell lines by
compounds 42, 44, 48, 62 and 69.
a
Compd
IC ± SD (nM)
50
U343MG
U87MG
T98G
SK-N-BE
SK-N-BE(2)-C
4
4
4
6
6
2
4
8
2
9
69 ± 5
30 ± 4
12 ± 2
20 ± 2
22 ± 2
61 ± 7
30 ± 0.4
31 ± 0.7
46 ± 4
54 ± 4
35 ± 3
37 ± 5
22 ± 3
24 ± 2
244 ± 1
311 ± 1
221 ± 1
288 ± 1
303 ± 1
106 ± 1
132 ± 1
56 ± 1
178 ± 1
147 ±1
48 ± 5
a
Cytotoxic concentrations for the indicated cell lines. Experiments were performed in triplicate.
Incubation time was 48 h. For U343MG cells incubation time was 72 h.
3
.5. Inhibition of growth of colorectal HT29, HCT116, SW480 and SW620 cell lines. Colorectal
cancer (CRC) is a commonly occurring cancer worldwide, and 95% of these tumors are
adenocarcinomas [29]. Early CRC cases are treated by surgery, but often the disease is diagnosed
at an advanced stage and sometimes with distant metastases. Adjuvant chemotherapy is needed,
but drug resistance may emerge, leading to failure of treatment [30]. Compounds 42, 44, 48, 62
and 69 showed strong inhibition of four CRC cell lines [HT29 (colorectal adenocarcinoma),
HCT116 (colorectal carcinoma), SW480 (colorectal adenocarcinoma) SW620 (colorectal
adenocarcinoma)] (Table 6). The IC values obtained ranged from 27 (48) in HCT116 cells, to
5
0
1
67 (62) nM in SW480 cells. ARDAP 48 was the most potent inhibitor of HCT116, SW480 and
SW620 cell lines, whereas 69 was the most active in the HT29 cells.

Table 6
Growth inhibition of HT29, HCT116, SW480, SW620 and T24 cell lines by compounds 42, 44, 48, 62
and 69
a
Compd
IC ± SD (nM)
50
HCT116
HT29
SW480
SW620
T24
4
4
4
6
6
2
4
8
2
9
42 ± 2
44 ± 2
27 ± 2
36 ± 2
41 ± 5
56 ± 1
51 ± 2
51 ± 2
35 ± 2
34 ± 1
162 ± 1
164 ± 1
48 ± 1
123 ± 1
38 ± 1
28 ± 1
47 ± 1
61 ± 1
34 ± 1
12 ± 2
12 ± 1
48 ± 1
58 ± 1
167 ± 4
153 ± 1
a
Cytotoxic concentrations for the indicated cell lines. Experiments were performed in triplicate.
Incubation time was 48 h.
3
.6. Inhibition of growth of urinary bladder T24 and ovary carcinoma ES-2 Cells. Compounds
2, 44, 48, 62 and 69 inhibited the growth of urinary bladder T24 cancer cells with IC ’s in the
4
5
0
nanomolar range ARDAPs 44 and 48 (IC of 12 nM in both cell lines) showed the strongest
5
0
antitumor activity (Table 6). The highly potent ARDAP 48 was evaluated as an inhibitor of
human ovary carcinoma ES-2 cells. In this assay 48 showed IC of 118 nM.
5
0
3
.7. Drug-like properties of compounds 42, 44, 48, 62 and 69 were predicted through the most
common descriptors of drug-likeness. The physical and chemical descriptors of the Lipinski [31]
Veber [32] and Egan [33] rules were computed to evaluate the bioavailability after oral
administration of derivatives 42, 44, 48, 62 and 69. There was no violation of the rules for these
five compounds, so they were predicted to have a good oral bioavailability (Table 7). The data of
derivative 48 are summarized in the radial graph (Fig. 2).

Table 7
Physico-chemical profiles of compounds 42, 44, 48, 62 and 69.
a,b
a,c
a,d
a,e
a,f
a,g
a,h
a,i
a,j
k,l
k,m
k.n
Comp Mw
H don
H acc
logP
logS
tPS
Lr
Vr
Er
Caco
MDCK
Khsa
42
44
48
62
69
446.47
473.48
443.50
446.47
443.50
2
3
4
2
4
6
9
7
6
7
4.62
4.35
3.84
4.62
3.84
-5.36
-5.28
-4.84
-5.36
75.7
121.5
101.7
75.7
0
0
0
0
0
0
0
0
0
0
0
1276
228
932
100
0.99
0.9
0
0
0
0
323
145
0.71
0.99
0.71
1277
323
1165
145
-4.84 101,73
a
b
c
d
e
f
Physicochemical properties predicted by FAF-drug4 server [34,35].
Molecular weight.
Number of H-bond acceptors.
Number of H-bond donors.
Octanol-water partition coefficient predictor by XLOGP3 method [36].
Logarithm of compound water solubility by ESOL method [37].
Topological polar surface area.
g
h
h
j
Lr: Lipinsky Rule deviation (log P <5, H-bond donors
≤5, H-bond acceptors
≤10, and a molecular weight <500) [31].
Vr: Veber rule deviation (rotatable bonds 10, tPSA
≤
≤140) [32].
Er: good/bad bioavailability (0≥ tPSA ≤132 and -1≥ logP ≤6) [33]
Physicochemical properties predicted by QikProp [38].
Caco: Apparent Caco-2 permeability (nm/sec) (<25 poor, >500 great).
k
l
m
n
MDCK - Apparent MDCK permeability (nm/sec) (<25 poor, >500 great). Logarithm of the predicted binding to human
serum albumin (-1.5 – 1.5).

Fig 2. A radial plot representing the computed oral bioavailability profile of 48. Structural properties of
compounds are compared with the optimal blue area defined from Rule-of-five and Veber’s rules.
Compound values (blue line) should fall within the green area; the red one, being an extreme zone,
generally indicates low oral bioavailability. The computations involved logP, molecular weight (MW),
topological polar surface area (tPSA), rotatable bonds (RotB), H-bond acceptors and donors (HBA,
HBD).
3
.8. Metabolic stability of compound 48. Aniline compounds may potentially cause
methemoglobin formation and hemolysis as their most prominent adverse effects. In particular,
the N-hydroxylation of aniline is responsible for oxidation of Fe(II) of oxyhemoglobin to Fe(III).
Hemoglobin-Fe(III) is a weak carrier of oxygen to tissues and leads to methemoglobinemia, low
levels of oxygen in the blood and red blood cell damage. Oxidation of the amino group of the
aniline decreases the amount of hemoglobin binding and increases mutagenicity and
carcinogenicity [39]. While several aniline compounds may undergo oxidation, there are also

many examples of drugs with aniline substructures that escape oxidative metabolism. Since the
most active derivatives reported in this work share aniline moieties, we evaluated in silico the
metabolic reactivity of these groups. Using the P450 module from the Schrödinger suite [40], we
computed the intrinsic reactivity of all atoms of the derivative 48 to the 3A4 Cyp isoform. The
carbon atoms of the methoxy groups and the unsubstituted aromatic carbon atoms of the
trimethoxyphenyl moiety showed higher likelihood of reactivity than the aniline nitrogen atoms,
which were predicted to have a low likelihood of oxidation. The computational prediction
suggests negligible metabolic oxidation of the aniline groups of 48. Further development of these
agents will include aniline group masking by their inclusion in heterocyclic rings or carboxamide
groups.
3
.9. In vivo antitumor activity of compound 48. Tumors could be observed on the backs of the
mice (injected T24 or ES-2) in the saline treatment group on Day 22, whereas no tumors were
found on the backs of the 48-treated nude mouse group. Mice were euthanized on Day 40, and
tumors on the backs were collected for measurement of tumor volume and weight. Results
showed that the tumors from the group treated with 48 were significantly smaller than those
obtained from the control group (saline treatment) in terms of both tumor volume and weight
(
Fig- 3). Meanwhile, hematoxylin and eosin (HE) staining showed that the type of tumor formed
in the two groups were human bladder transitional cell carcinoma (from T24-injected mice) or
human ovarian clear cell carcinoma (from ES-2-injected mice) (Figure 5SD, supplementary
data).

Fig. 3. A. Mice injected with T24 (top panel) or ES-2 (bottom panel) tumors after 40 days of treatment with
compound 48 or with saline. B. Tumor weight at day 40. C. Tumor size at day 40.
Immunofluorescence staining results showed that the tumors from the 48-treated group (ES-2
or T24) had significantly lower expression of the proliferation factor Ki67, a marker for tumor
angiogenesis CD31, a mitochondrial stability protein Bcl-2, compared with the control group
(
Fig. 4). In addition, the tumors from the untreated group (ES-2 or T24) had significantly higher
expression of apoptotic factors caspase 9 and caspase 3 and a mitochondrial disintegration