Benzimidazolyl-pyrazolo[3,4- b]pyridinones, Selective Inhibitors of MOLT-4 Leukemia Cell Growth and Sea Urchin Embryo Spiculogenesis: Target Quest

Article abstract of DOI:10.1021/acscombsci.9b00135

1,3-Substituted pyrazolo[3,4-b]pyridinones 11-18 were synthesized by a three-component condensation of Meldrum's acid with aryl aldehydes and 1,3-substituted 5-aminopyrazoles. Their biological activity was evaluated using the in vivo phenotypic sea urchin embryo assay and the in vitro cytotoxicity screen against human cancer cell lines. In the sea urchin embryo model, 1-benzimidazolyl-pyrazolo[3,4-b]pyridinones 11 caused inhibition of hatching and spiculogenesis at sub-micromolar concentrations. These compounds also selectively and potently inhibited growth of the MOLT-4 leukemia cell line. Subsequent structure-activity relationship studies determined the benzimidazolyl fragment as an essential pharmacophore for both effects. We applied numerous techniques for target identification. A preliminary QSAR target identification search did not result in tangible leads. Attempts to prepare a relevant photoaffinity probe that retained potency in both assays were not successful. Compounds 11 were further characterized for their activity in a wild-type versus Notch-mutant leukemia cell lines, and in in vitro panels of kinases and matrix metalloproteinases. Using a series of diverse modulators of spiculogenesis as standards, we excluded multiple signaling networks including Notch, Wnt/β-catenin, receptor tyrosine kinases (VEGF/VEGFR, FGF/FGFR), PI3K, and Raf-MEK-ERK as possible targets of 11. On the other hand, matrix metalloproteinase-9/hatching enzyme was identified as one potential target.

Full text of DOI:10.1021/acscombsci.9b00135

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Article
Benzimidazolyl-pyrazolo[3,4-b]pyridinones - selective inhibitors of MOLT-4
leukemia cell growth and sea urchin embryo spiculogenesis: Target quest.
Boris V. Lichitsky, Andrey N. Komogortsev, Arkady A. Dudinov, Mikhail M Krayushkin, Evgenii N.
Khodot, Alexander V. Samet, Eugenia Silyanova, Leonid D. Konyushkin, Alexei S Karpov, Delphine
Gorses, Thomas Radimerski, Marina N. Semenova, Alex S. Kiselyov, and Victor Vladimirovich Semenov
ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.9b00135 • Publication Date (Web): 05 Nov 2019
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Benzimidazolyl-pyrazolo[3,4-b]pyridinones - selective inhibitors of MOLT-4 leukemia cell
growth and sea urchin embryo spiculogenesis: Target quest
Boris V. Lichitsky,† Andrey N. Komogortsev,† Arkady A. Dudinov,† Mikhail M. Krayushkin,†
Evgenii N. Khodot,† Alexander V. Samet,† Eugenia A. Silyanova,† Leonid D. Konyushkin,† Alexei
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S. Karpov,‡ Delphine Gorses,‡ Thomas Radimerski,‡ Marina N. Semenova,§ Alex S. Kiselyov,‖
Victor V. Semenov*,†
† N. D. Zelinsky Institute of Organic Chemistry, RAS, Leninsky Prospect, 47, 119991, Moscow,
Russian Federation
‡ Novartis Institutes for BioMedical Research, CH-4056 Basel, Switzerland
§ N. K. Kol’tsov Institute of Developmental Biology, RAS, Vavilov Street, 26, 119334, Moscow,
Russian Federation
‖ Myocea, Inc., 9833 Pacific Heights Blvd., San Diego, CA 92121, USA
Corresponding author: Victor V. Semenov
Address: N. D. Zelinsky Institute of Organic Chemistry, RAS, Leninsky Prospect, 47, 119991,
Moscow, Russian Federation. Tel.: +7 499 135 6343; fax: +7 499 137 2966.
E-mail: vs@zelinsky.ru
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ABSTRACT
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1,3-Substituted pyrazolo[3,4-b]pyridinones 11–18 were synthesized by a three-component
condensation of Meldrum’s acid with aryl aldehydes and 1,3-substituted 5-aminopyrazoles. Their
biological activity was evaluated using the in vivo phenotypic sea urchin embryo assay and the in
vitro cytotoxicity screen against human cancer cell lines. In the sea urchin embryo model, 1-
benzimidazolyl-pyrazolo[3,4-b]pyridinones 11 caused inhibition of hatching and spiculogenesis at
submicromolar concentrations. These compounds also selectively and potently inhibited growth of
the MOLT-4 leukemia cell line. Subsequent structure-activity relationship studies determined the
benzimidazolyl fragment as an essential pharmacophore for both effects. We applied numerous
techniques for the target identification. A preliminary QSAR target identification search did not
result in tangible leads. Attempts to prepare a relevant photoaffinity probe that retained potency in
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both assays were not successful. Compounds 11 were further characterized for their activity in a wild
type vs Notch-mutant leukemia cell lines, and in in vitro panels of kinases and matrix
metalloproteinases. Using a series of diverse modulators of spiculogenesis as standards, we excluded
multiple signaling networks including Notch, Wnt/-catenin, receptor tyrosine kinases
(VEGF/VEGFR, FGF/FGFR), PI3K, and Raf-MEK-ERK as possible targets of 11. On the other
hand, matrix metalloproteinase-9/hatching enzyme was identified as one potential target.
KEYWORDS: benzimidazolyl-pyrazolo[3,4-b]pyridinone, cytotoxicity, MOLT-4, sea urchin
embryo, spiculogenesis
INTRODUCTION
Multicomponent domino reactions are versatile tools for expeditious access to a variety of
biologically active compounds. For instance, a three-component condensation of Meldrum’s acid 1
with aromatic aldehydes 2 and 5-membered aminoheterocycles A (amino-imidazoles, -pyrazoles, -
thiazoles, isothiazoles, thiophenes) furnishes diverse dihydropyridones B (Figure 1).1 In a further
development of this protocol, we introduced a facile synthesis of 3,7-diaryl-6,7-dihydroisothiazolo-
[4,5-b]pyridin-5(4H)-ones C. Gratifyingly, multiple derivatives from these series including
compound C1 (Figure 1) were identified to be potent antimitotics with microtubule destabilizing
mode of action, as demonstrated by the in vivo phenotypic sea urchin embryo model.2 Furthermore,
selected compounds displayed a significant cytotoxicity against human cancer cells including
multidrug resistant cell lines with GI50 values less than 1 M.2
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R1
R2
R2
R2
A
R2
A
O
O
O
O
R1
x
R3
R4
R1
R3
R4
R1
R3
R4
A
R4
R
A
A
R3
2
O
O
NH2
S
B
S
B
x
B
B
N
R5
y
N
N
N
B
z
O
O
+
y
z
N
H
O
N
H
O
N
H
N
H
O
R
Ar
1
A
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R = H, Alk, Ar
x, y, z = C, N, S
R1-R4 = H, Alk, OAlk
B
C
C1
D
H3CO
R5 = H, Alk, Ar, Het
Figure 1. Reaction scheme for azolopyridinones.
In the ongoing search for the novel tubulin destabilizing agents, we turned our attention to a
chemotype B, and the respective pyrazolo[3,4-b]pyridinones D in particular. The synthesis of
pyrazolopyridinone derivatives D has been explored recently.3-7 Roshan, et al. reported a one-pot,
three-component reaction to access novel derivatives of pyrazolo[3,4-b]pyridine-6(7H)-ones (Figure
1, D, R5 = H,) with excellent yields (87%–95%) from 5-amino-3-methyl-1H-pyrazole.3 Several
analogues exhibiting general structure D displayed a diverse spectrum of biological activities
including antimicrobial,4, 8 antiviral,9 anti-inflammatory,10 GSK3-inhibiting,11 and proapoptotic8
effects. In a recent publication, these molecules were identified as agonists of the G-protein coupled
receptor 39 (GPR39).12
Encouraged by these data, we synthesized a series of compounds represented by the general
scaffold D with diverse substituents R1–R4 in the aromatic ring A and R5 in the pyrazole ring B.
Biological activities, structure-activity relationship (SAR), and possible mechanism(s) of action of
these targeted compounds were evaluated using a phenotypic sea urchin embryo model, human
cancer cell lines, and biochemical target-based screen.
RESULTS AND DISCUSSION
Chemistry. 1,3-Substituted pyrazolo[3,4-b]pyridine-6(7H)-ones 11–18 were synthesized using
three-component condensation of Meldrum’s acid 1, aryl aldehydes 2, and 1,3-substituted 5-
aminopyrazoles 3–10 (Scheme 1).
Scheme 1. Synthesis of pyrazolo[3,4-b]pyridinones 11–18.
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R3
A
R4
R6
R2
R1
R6
R4
R3
O
O
O
AcOH
A
N
B
N
B
+
+
R1
O
O
N
reflux, 2h
NH2
N
O
NH
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R2
2a-v
R3
R5
R5
11a-u; 12j; 13b,j; 14d,f,g;
15b,j; 16c,j,o; 17v; 18b,j
1
3-10
R1
R2,
R4
a:
H
H
H
H
H
H
H
H
H
OH
OMe
H
OH
OMe
OMe
H
H
H
H
H
H
H
b:
c:
d:
e:
f:
Cmpd
R5
R6
OMe
OMe
OH
N
NH
3, 11:
Me
OMe
g:
-OCH2O-
4, 12:
5, 13:
Me
Me
Me
H
h:
i:
H
OMe
OMe OMe
H
H
OH
OMe
OMe
H
OMe
OMe
j:
k:
l:
H
H
H
OMe
OMe
OMe
6, 14:
7, 15:
Me
Me
-OCH2O-
OMe
-OCH2O-
MeO
m:
n:
OMe OMe
OMe
OMe
N
OMe
N
o:
p:
q:
r:
OMe OMe
-OCH2O-
Cl
H
H
OMe OCH2CONH2
H
H
H
H
OCH2-4F-C6H4
OCH2-2Cl-C6H4
H
H
8, 16:
H
O
O
O
H3CO
NH
s:
N
A
S
9, 17:
H
H
t:
H
H
H
OMe
OMe
OMe
OC7H15
OC9H19
OEt
OMe
OMe
H
u:
v:
10, 18:
H
In the modified procedure to make benzimidazolyl-pyrazolo[3,4-b]pyridinones (BIPP) 11a–u, we
improved both feasibility and yield of the key intermediate, 2-hydrazinyl-1H-benzimidazole 21
(Scheme 2, A). Specifically, starting benzimidazole-2-thione 19 was oxidized easily with aqueous
sodium percarbonate to afford 1H-benzimidazole-2-sulfonic acid 20. Further conversion of 20 into
21 was accomplished in the aqueous N2H4 at moderate pressure under heating. Compound 21 was
further reacted with cyanoacetone to afford the intermediate 1-(1H-benzimidazol-2-yl)-3-methyl-1H-
pyrazol-5-amine 3 followed by its three-component condensation to furnish the desired derivatives
11a–u (Scheme 2, B, 49–82% overall yields). Further modifications of the benzimidazole
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pharmacophore were accomplished via base-mediated alkylation of the NH to result in 11bx, 11by,
11jx, 11jy, and 11jz.
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Scheme 2. Synthesis of benzimidazolyl-pyrazolo[3,4-b]pyridinones 11 (BIPP).
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N
O
S
OH
O
A
N
N
NH2
NH
NH
NH2
S
N
N
N
HN
.
, EtOH
N2H4
Na2CO3 1.5H2O2
O
NH
NH
NH
60-75 oC, 40 min
120
°
C, 4 h
reflux, 12 h
19
20
21
3
64%
84%
R3
53%
R3
B
R4
R2
R4
R2
R1
N
R7Hal
N
NH2
R4
O
O
N
O
K2CO3, DMF
AcOH
N
N
NH
+
+
R3
O
O
N
N
R1
reflux, 2h
rt, 3h
N
O
N
NH
R7
O
NH
N
R2
NH
3
1
2a-u
11a-u
11bx,by,jx,jy,jz
R2,
H
R3
R4
R7
bx:
OMe
OMe
H
H
CH3
by:
jx:
jy:
jz:
H
C7H15
CH3
OMe OH OMe
OMe OH OMe
OMe OH OMe
C7H15
N
N
Phenotypic Sea Urchin Embryo Tests. Considering the reported antimitotic microtubule
destabilizing activity of isothiazolopyridinones C (Figure 1),2 we decided to evaluate related
pyrazolopyridinones 11–18 for their antitubulin activity using a phenotypic sea urchin embryo assay.
The assay was developed and extensively validated by our group over the past decade. It provides
data on the specific antimitotic microtubule destabilizing activity related to several particular steps of
differentiation and morphogenesis of the embryo.13, 14 Particularly, cleavage (cell division)
alteration/arrest after fertilized egg treatment evidences antimitotics activity. Specific changes of
embryo motility after treatment of hatched blastulae including spinning at the bottom of the vessel
instead of forward swimming near the seawater surface evidences microtubule destabilizing mode of
action. The effects of compounds 11–18 on the sea urchin embryos are summarized in Tables 1 and
2. A close analogue of 14g, isothiazolopyridinone C1 (Figure 1),2 and natural antimitotic
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combretastatin A4 disodium phosphate (CA4P) served as reference compounds. In contrast to
isothiazolopyridinones,2 only four molecules were characterized as antimitotic antitubulin agents.
Specifically, pyrazolopyridinones 14g and 16o induced cleavage alteration/arrest and embryo
spinning, whereas 14d and 17v triggered the formation of tuberculate-shaped arrested eggs, both
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effects are suggestive of the microtubule destabilizing activity. Notably, pyrazolopyridinone 14g was
less active than its isothiazole analog C1. The majority of pyrazolopyridinones caused neither
cleavage arrest with tuberculate eggs, nor embryo spinning, the phenotypic effects directly linked to
microtubule destabilization. Compounds 11e, 11m–p, 14f, 15b, 16c, and 18b exhibited modest
antiproliferative activity altering cleavage at relatively high concentrations (1–4 M). However, it
should be pointed out that, as opposed to tubulin/microtubule targeting compounds, numerous
benzimidazolyl-pyrazolopyridinones (BIPP) 11, namely, 11a–q, and 11s, selectively inhibited
spiculogenesis, a formation of the embryonic skeleton (Figure 2A), with good potency (EC values of
0.05–1 M, Table 1). Most of these compounds, specifically, 11a, 11e–j, 11k, 11l, 11o, 11p, and
11s, also inhibited hatching at concentrations of 0.5–4 M, higher than those necessary for spicule
impairments (Figure 2B). These two well-known phenotypic effects that are not exhibited by
antitubulin agents were readily observed and are likely to be related to distinct developmental
processes discussed below.
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Figure 2. Typical effects of benzimidazolyl-pyrazolopyridinones (BIPP) 11 on the sea urchin
embryos as exemplified by 11j (A, 30 h postfertilization) and 11o (B, 12 h postfertilization).
Compounds were added to fertilized eggs. (A) 11j (0.2 and 0.5 M) selectively inhibited
spiculogenesis. At 0.2 M concentration spicule rudiments (arrowheads) are visible in some
embryos. Control: Intact two-arm plutei. (B) Control: Intact hatched mesenchyme blastula with
primary mesenchyme cells (PMC) inside blastocoel. 11o (4 M) caused hatching arrest, and
mesenchyme blastula developed inside fertilization envelope (arrowhead). Incubation temperature:
24 °C. Scale bars: 100 m.
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Table 1. Effects of Benzimidazolyl-pyrazolopyridinones 11 on MOLT-4 Cells and Sea Urchin Embryos
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2
3
4
5
6
7
8
sea urchin embryo effects, EC (M)a
cleavage hatching spicule
alteration inhibition alteration
compd
NCI60 screen, SRB assay
resazurin assay
no
spicules
growth %c,
MOLT-4/mean
for 60 cell lines
-14.44/80.04
GI50 (M)b,
MOLT-4/mean
for 60 cell lines
NAd
MOLT-4 GI50 (M)b
(mean ± SE)
11a
11b
11c
11d
11e
11f
>4
>4
>4
>4
4
2
>4
4
0.2
0.2
2
0.5
>4
>4
0.2
0.2
0.5
4
0.11 ± 0.01
0.17 ± 0.03
0.21 ± 0.03
0.11 ± 0.01
0.08 ± 0.01
0.12 ± 0.01
0.20 ± 0.03
0.20 ± 0.03
0.12 ± 0.01
0.16 ± 0.01
0.26 ± 0.02
0.12 ± 0.01
0.23 ± 0.05
NAd
9
0.0657/2.69
NAd
-44.57/77.08
NAd
10
11
12
13
14
15
16
17
18
19
20
21
22
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1
0.05
0.05
0.2
1
NAd
NAd
0.5
4
NAd
NAd
>4
>4
>4
>4
>4
>4
>4
1
0.0405/4.27
0.0367/2.34
NAd
-31.49/63.48
-48.45/28.61
NAd
11g
11h
11i
2
2
0.1
1
2
4
4
0.0478/3.89
0.0427/2.75
NAd
-30.97/62.58
-36.03/44.18
-42.49/96.79
NAd
11j
2
0.05
0.2
0.1
0.1
0.1
0.1
0.1
0.1
4
0.2
0.5
4
11k
11l
4
1
NAd
11m
11n
11o
11p
11q
11r
11s
11t
>0.5
>0.5
1
2
NAd
NAd
1
2
NAd
NAd
4
4
NAd
NAd
NAd
4
2
0.5
1
NAd
NAd
0.12 ± 0.01
4.1 ± 0.5
NAd
>4
>4
>4
>4
>4
>4
2
NAd
7.48/60.01
57.24/84.68
NAd
>4
0.5
>4
NAd
0.1
2
NAd
NAd
>4
NAd
NAd
6.4 ± 1.6
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11u
11bx
11by
11jx
11jy
11jz
C12
>4
>4
>4
>4
4
>4
>4
>4
4
>4
>4
NAd
NAd
NAd
NAd
NAd
1
2
3
4
5
6
7
8
NAd
NAd
>4
>4
>4
NAd
NAd
>4
>4
>4
NAd
NAd
4.5 ± 0.9
>10
>4
>4
>4
NAd
NAd
9
>4
>4
>4
NAd
>4
NAd
NAd
NAd
10
11
12
13
14
15
16
17
18
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0.01
NAd
NAd
1.29/1.55
81.63/77.34
NAd
a The sea urchin embryo assay was conducted as described previously.13, 14 Fertilized eggs and hatched blastulae were exposed to 2-fold decreasing
concentrations of compounds. Duplicate measurements showed no differences in effective threshold concentration (EC) values. No cleavage arrest and
embryo spinning were observed. b GI50: concentration required for 50% cell growth inhibition. c Cell growth at 10 M concentration. d NA: not available.
Table 2. Effects of Pyrazolo[3,4-b]pyridinones 12–18 on MOLT-4 Cells and Sea Urchin Embryos
sea urchin embryo effects, EC (M)a
cleavage
alteration
compd
NCI60 screen, SRB assay
resazurin assay
MOLT-4 GI50
(M)b
cleavage
arrest
embryo
spinning
spicule
alteration
growth %c,
MOLT-4/mean
for 60 cell lines
103.38/96.99
NAd
GI50 (M)b,
MOLT-4/mean for
60 cell lines
NAd
12j
13b
13j
14d
14f
14g
15b
15j
16c
>4
>4
>4
0.5
4
>4
>4
>4
>4
>4
>10
>4
2
>4
>4
>10
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
>4
>4
NAd
2 TEe
>4
NAd
>4
NAd
NAd
0.05
2
0.5
>4
NAd
>4
NAd
>4
>4
>4
NAd
>4
2
>4
>4
NAd
>4
>4
127.27/101.63
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16j
16o
>4
0.2
>4
1
>4
4
>4
NAd
NAd
>4
NAd
0.600/0.562
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
1
2
3
4
5
6
7
8
1.51/19.05
NAd
17v
0.5
1 TEe
>4
>5
>4
>4
0.2
0.05
18b
4
NAd
NAd
18j
>4
>4
>4
NAd
97.59/97.77
81.63/77.34
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9
C12
0.01
0.005
0.1
0.01
NAd
NAd
1.29/1.55
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CA4P2
a The sea urchin embryo assay was conducted as described previously.13, 14 Fertilized eggs and hatched blastulae were exposed to 2-fold decreasing
concentrations of compounds. Duplicate measurements showed no differences in effective threshold concentration (EC) values. No hatching inhibition was
observed. b GI50: concentration required for 50% cell growth inhibition. c Cell growth at 10 M concentration. d NA: not available. e TE: tuberculate eggs
typical of microtubule destabilizing agents. f NCI60 screen data for NSC #645646, combretastatin A-4 disodium phosphate (CA4P).
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The structure-activity relationship (SAR) analysis suggested the importance of the benzimidazole
fragment for the observed specific sea urchin embryo effects. Namely, compounds 12–18 lacking
benzimidazole moiety, 12j, 13b, 13j, 14f, 15b, 15j, 16c, 16j, 18b, and 18j, did not induce either specific
inhibition of hatching or spicule formation (Tables 1 and 2). Interestingly, replacement of the
benzimidazole group changed biological activity of the resulting derivatives in the sea urchin embryo
assay. For example, molecules 14d, 14g, 16o, and 17v were identified as microtubule destabilizing agents.
These data were in contrast to the outcome shown by respective benzimidazole-containing analogues 11d,
11g, 11o, and 11f that inhibited hatching and spiculogenesis. Substitution at the N atom of the
benzamidazole fragment (11bx, 11by, 11jx, and 11jy) caused activity loss, as compared to 11b and 11j.
Alkylation of the 4-OH pharmacophore in the aromatic ring A also yielded inactive compounds 11t and
11u. We did not observe any SAR correlation between the rate of hatching delay/arrest and suppression of
spicule formation, suggesting possible interaction of BIPP 11 with more than one cellular target.
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Cytotoxicity Against Human Cancer Cells. Compounds 11a, 11b, 11f, 11g, 11i–k, 11q, 11r, 12j, 16c,
16o, and 18j were tested in the NCI60 screen to assess cytotoxicity against 60 human cancer cell lines
using sulforhodamine B (SRB) assay. The results are presented in Tables 1 and 2. Molecules 11b, 11f,
11g, 11i, and 11j containing substituted benzimidazole fragment were tested at five doses. Interestingly,
they were found to selectively and potently inhibit growth of MOLT-4 human T-cell acute lymphoblastic
leukemia cells with GI50 < 0.1 M (Table 1, Supplementary Figures S1–S10). Notably, the compounds
modulated growth of MOLT-4 cells in a dose dependent fashion with growth inhibition reaching maximum
at 1 M concentration. This MOLT-4 specificity was not observed at a higher concentrations (10–100 M)
as demonstrated in Supplementary Figures S1, S3, S5, S7, S9, and S11–S15, with the exception of 11k that
exhibited selectivity against MOLT-4 cells at 10 M concentration (Table 1, Supplementary Figure S16).
BIPP 11a, 11q, and 11r tested at 10 M concentrations also didn’t display selectivity against MOLT-4
cells (Supplementary Figures S17–S19). Replacement of benzimidazole in 11j with methyl group yielded
inactive compound 12j (Table 2). Similarly, related derivatives featuring (furan-2-yl)methylimidazole ring
B showed neither cytotoxicity at up to 10 M concentration (16c), nor selectivity against MOLT-4 cells
(16o, Table 2, Supplementary Figures S20 and S21). Compound 18j with unsubstituted imidazole ring B
was inactive as well (Table 2).
In the next step, we tested a set of BIPP (11a–m, 11p, 11q and 11t) and compound 12j for MOLT-
4 cell viability/proliferation using resazurin assay selected due to its validation, reliability and high
dynamic range.15, 16 In this test system, molecules 11 containing the benzimidazole fragment displayed
high activity with GI50 values of 0.077–0.257 M (Table 1) except for 11q and 11t with bulky substituents
in the aromatic ring A (GI50 = 4.1 ± 0.5 and 6.4 ± 1.6 M, respectively). 12j lacking the benzimidazole
fragment was inactive.
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Thus far, cytotoxicity SAR studies revealed that the benzimidazole pharmacophore attached to the
pyrazolopyridinone template as well as the hydroxy- and/or methoxy-substituted phenyl ring A were both
essential for the selective MOLT-4 cells growth inhibition. Neither the number of hydroxy/methoxy
substitutients nor their position in the phenyl ring A influenced this activity. On the contrary, introduction
of bulky substituents into the ring A (compounds 11q, 11r, and 11t) decreased the respective cytotoxicity.
Importantly, SAR analysis showed good correlation between selective cytotoxicity against MOLT-
4 cells and phenotypic sea urchin embryo data. Potent and selective inhibitors of MOLT-4 cell growth,
BIPP 11a–m and 11p also caused hatching inhibition and specifically altered spiculogenesis in sea urchin
embryos (Table 1). Compounds lacking benzimidazole moiety, 12j, 16c, and 18j were not selective against
MOLT-4 cells. These molecules did not induce specific inhibition of hatching or spicule formation (Table
2). A weak cytotoxic agent 11r modestly modulated spicule growth in the sea urchin embryo. It is likely
that the replacement of the benzimidazole group affected biological activity of the resulting derivatives in
both assays. For example, molecule 16o induced cleavage alteration/arrest and the embryo spinning, both
suggestive of its microtubule destabilizing activity. In the NCI60 screen, this compound showed
cytotoxicity (mean GI50 = 0.562 M) without specific inhibition of MOLT-4 cell growth typical for
tubulin/microtubule targeting agents (for example, see NCI cancer screen data for combretastatin A-4,
NSC # 613729, https://dtp.cancer.gov/dtpstandard/cancerscreeningdata/index.jsp). Our SAR data
suggested that specific inhibition of both MOLT-4 cell growth and the sea urchin embryo spiculogenesis
caused by BIPP 11 may be mediated by the same set of molecular targets or their sea urchin orthologues.
Furthermore, in vivo sea urchin embryo assay data led us to conclude that cancer cell growth inhibition
caused by BIPP 11 was not related to the microtubule destabilizing activity. Thus, in the next step we
attempted to study possible cell target(s) and mechanism of action for the lead series 11.
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Search for a Possible Cellular Target of BIPP 11. In the initial approach, we analyzed the ChEMBL
bioactivity database in order to identify related structures and link them to a specific molecular target(s).
Unfortunately, the query yielded a single match, namely the compound 11a. Whereas the molecule showed
activity against parasitic protozoans (kinetoplastids), its IC50 values against THP-1 human monocytic
leukemia cells and HepG2 human liver carcinoma cells were 6.31 and 25.12 M, respectively
(http://dx.doi.org/10.6019/CHEMBL3430912). Neither mode of action nor cellular target data were
available from the literature.
Considering that MOLT-4 is a Notch mutant cell line, we next conducted cytotoxicity studies of
compound 11j on 7 wild type (wt) and 4 Notch-mutant leukemia cell lines (Table 3). The tests showed no
preferential inhibition of Notch mutants compared to MOLT-4 cell line. Specifically, three Notch-mutant
leukemia cell lines as well as all wt lines were much less sensitive towards 11j. Compound 12j featuring
methyl group instead of benzimidazole functionality was inactive. These results correlated well with the
respective NCI60 screen data (Table 3) suggesting that the series of interest do not affect Notch signaling.
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Table 3. Cytotoxicity of 11j Against Wild Type (wt) and Notch-mutant (mut) Leukemia Cells
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5
6
7
8
cell line
origin
Notch mutation
resazurin assay
GI50 (M) (mean ± SE)
SUP-T11
LOUCY
Jeco-1
Mino
MOLT-16
HL-60
T-ALLa
T-ALLa
B-cell lymphoma
B-cell lymphoma
T-ALLa
wt
wt
wt
wt
wt
wt
wt
mut
mut
mut
mut
2.20 ± 0.00
3.65 ± 0.05
>10
5.7 ±0.9
7.4 ± 0.8
>10
1.5 ± 0.3
0.14 ± 0.01
2.70 ± 0.06
>10
9
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60
promyelocytic leukemia
Jurkat
T-ALLa
MOLT-4
HPB-ALL
RPMI-8402
Rec-1
T-ALLa
T-ALLa
T-ALLa
B-cell lymphoma
NCI60 screen, SRB assay
acute myeloid leukemia
6.5 ± 0.2
HL-60 (TB)
K-562
CCRF-CEM
MOLT-4
wt
wt
mut
mut
2.82
3.98
1.47
chronic myeloid leukemia
T-ALLa
T-ALLa
0.0427
aT-ALL: T-cell acute lymphoblastic leukemia
We next attempted photoaffinity labeling to identify the molecular target(s) of BIPP 11.17 The key
challenges to address when applying this approach included selection of a) a suitable linker and b) feasible
attachment site to maintain biological activity of the resulting small molecule following photoactivation
and covalent linking. In our study, nitrogen atom of the benzimidazole fragment (Scheme 2, R7) and 4-OH
group in the phenyl ring A (Scheme 1, R3, t and u) were prioritized as potential connecting points,
primarily due to the results from the SAR studies and relative ease of their chemical modification with
photoreactive group. Compounds 11t and 11u, tentative analogues of 11j containing O-alkyl groups, were
synthesized from the corresponding aromatic aldehydes 2t and 2u (Scheme 1). Molecules 11bx, 11by,
11jx, 11jy, and 11jz, substituted at the N atom of the benzamidazole fragment, were synthesized by
alkylation of the corresponding precursors 11b and 11j with alkyl halides (Scheme 2). Unfortunately, we
observed a considerable decrease in cytotoxicity of 11t, 11jx, and 11jy against MOLT-4 cells (Table 2)
even for the respective methyl derivative 11jx. The sea urchin embryo tests further confirmed that
modifications of the N atom in benzimidazole (11bx, 11by, 11jx, and 11jy) or 4-hydroxy group of the A
ring (11t and 11u) with potential linker reduced or eliminated the activity of compounds (Table 1). A
derivative of 11j modified with the diazirine PAL linker,18 11jz, (Scheme 2) didn’t show any phenotypic
effects in the sea urchin embryos (Table 1). In addition, modifications of the N atom of benzimidazole
dramatically reduced solubility of the derivatives. We concluded that the positions selected for
photoaffinity labeling were not suitable rendering the suggested photoaffinity labeling approach
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unacceptable for target identification. In our assessment, a more in-depth search for the alternative linker
attachment sites may require a significant investment into a rather involved and challenging chemistry.
The next attempt for target identification included QSAR virtual screening model19 via a free accessible
website http://rfqsar.kaist.ac.kr/home.php. This model is based on the ChEMBL dataset of human proteins.
The query results for 11j including the top-10 from overall 1,288 potential targets are presented in
Supplementary Figure S22. Kinases were found to be the most probable targets, FGFR1 leading the list
(probability factor of 0.28 or 28%). Therefore, in the next series of experiments we evaluated the effect of
11j on a panel of kinases (Supplementary Table S1). The panel comprised kinases associated with mitosis,
apoptosis, different signaling pathways, including those participating in regulation of spiculogenesis.
Unfortunately, the molecule did not display any notable inhibition of the receptor-type tyrosine-protein
kinases (AURKA, FGFR2, FGFR4, FLT3, and ZAP70), non-receptor tyrosine-protein kinases (ABL1,
ACVR1, BTK, JAK2, and Lyn), and serine/threonine-protein kinases (DAPK1, GSK3B, IRAK1, IRAK4,
MAP3K7, MAP4K4, PRKCA, PRKCQ, STK4, STK17A, and STK17B) up to 10 M concentration.
Whereas it is still possible the molecule binds to other targets in the expanded kinome, we felt the selected
panel was both sufficiently comprehensive and representative of all major kinase classes.
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Next, we evaluated compounds 11j and 12j in a panel of matrix metalloproteinases (MMPs). The
choice of MMPs was based on the ability of BIPP 11 to impair both hatching and spiculogenesis of the sea
urchin embryos. The sea urchin embryos hatch from fertilization envelope at mid-blastula. At this stage,
hatching enzyme (Zn-, Ca-dependent matrix metalloproteinase; envelyzin; EC 3.4.24.12)20, 21 digests the
fertilization envelope, and embryos start to swim. The majority of MOLT-4-selective BIPP 11 were found
to be hatching suppressors, suggesting their possible interaction with MMPs. Literature data indicate that
in addition to hatching regulation, MMPs in the sea urchin embryos participate in the primary mesenchyme
cells (PMC) fusion and syncytium formation necessary for spiculogenesis,22-24 as well as in the regulation
of spicule elongation.25 The target-based screen showed that 11j did not exhibit any appreciable inhibitory
activity against a set of MMPs, including MMP-1, 2, 8, 12, 13, 14, and ADAM17 (disintegrin and
metalloproteinase domain-containing protein 17). Intriguingly, 11j inhibited MMP-9 with the IC50 value of
ca. 490 nM, whereas 12j used as a negative control was inactive. Considering the above results, we
hypothesized that MMP-9 could be a molecular target. To verify the hypothesis, cytotoxicity of CGS
27023A (a reported inhibitor of MMP-1, MMP-2, MMP-3, and MMP-926) was tested in the MOLT-4 cells
to show no effect at concentrations up to 10 M. Moreover, a tetracycline antibiotic doxycycline known to
inhibit multiple MMP isoforms displayed average GI50 value of 3.05 M over 60 cell lines without any
selectivity against MOLT-4 cells (NCI60 screen data, NSC # 756751; https://dtp.cancer.gov). For
comparison, effects of four standard MMP inhibitors (GM6001, marimastat, CTS-1027, and T-5224) on
the sea urchin embryos were assessed (Supplementary Table S2). In our hands, all tested MMP inhibitors
failed to affect cleavage and/or embryo viability at concentrations up to 4 M. Multiproteinase inhibitors
GM6001 and marimastat both affected MMP-9, suppressed the embryo hatching at 0.02 M and 0.05 M,
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respectively and inhibited spicule formation at 0.2 M. Inhibitor of MMP-2 and MMP-13, CTS-1027, was
less potent altering hatching and spiculogenesis at 1 M and 4 M concentrations, respectively. T-5224
reported to inhibit MMP-3 and MMP-13 was inactive. Interestingly, the most potent agents GM6001 and
marimastat both reportedly target MMP-9 (https://www.medchemexpress.com), a MMP family member
that was also inhibited by 11j. However, in contrast to the reported MMP inhibitors, BIPP 11 altered
spiculogenesis at markedly lower concentrations than those suppressing embryo hatching (Table 1),
suggesting interaction with an additional molecular network(s) involved in the sea urchin embryo spicule
formation. Considering these observations, MMP/MMP-9 could be one of the targets of BIPP 11.
However, this mode of action is not likely to be related to the selective inhibition of MOLT-4 cell growth.
Since all attempts of BIPP 11 target(s) identification summarized above were unsuccessful, in the
next series of experiments we conducted a more detailed study focused on the specific impairment of
spicule formation caused by BIPP. Spiculogenesis of the sea urchin embryo (Supplementary Figure S23)
involves several steps including: 1) ingression of spiculogenic cells (PMCs, the only cells responsible for
spicule formation and growth) at a blastula stage, approximately at 1–2 h post-hatching; 2) directional
migration of PMC inside blastocoel by means of filopodia to afford two ventrolateral clusters during
gastrulation; 3) PMC fusion to form a syncytium, and 4) spicule development, including uptake of Ca2+
and Mg2+ ions from seawater and secretion of skeletal material (carbonate Ca, less carbonate Mg, and
spicule matrix proteins) into PMC syncytial cable via endocytosis.27 Considering this evidence from the
developmental biology, we selected a set of inhibitors that were likely to modulate signaling cascades and
proteins involved in the spicule formation, and their effects were compared to the phenotypic changes
caused by BIPP 11 (Figure 3, Supplementary Table S3). Specifically, we determined that when the control
blastulas hatched from the fertilization envelope and started to swim (Figure 2B and Figure 3a), zygotes
exposed to either BIPP (Figure 2B) or MMP inhibitors (Figure 3b) resulted in blastulas that continued to
develop inside the fertilization envelope for several hours. At latter stages of development BIPP selectively
inhibited spiculogenesis, but the embryos retained bilateral symmetry, normal motility and gut
differentiation (Figure 2A).
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Next, since mitogen-activated protein kinases (MAPK) were reported to play the key role in the sea
urchin embryo skeletogenesis,28-30 we tested several specific inhibitors of MAPKs (ex., MEK1/2, ERK1/2,
and Raf) (Supplementary Table S3). Multiple compounds caused selective concentration-dependent
suppression of spicule formation up to complete spicule elimination when added to the fertilized eggs
(Figure 3, panel e). However, as opposed to BIPP, they strongly inhibited ingression of the spiculogenic
PMC at a blastula stage (Figure 3c, Supplementary Table S3) and caused exogastrulation with
differentiated gut (Figure 3f, Supplementary Table S3) in accordance with the literature data. 28-30
Compounds that modulate transforming growth factor /transforming growth factor  receptor
(TGF/TGFR) signaling pathway effectively suppressed spiculogenesis, however they also triggered
radialization to produce a bell-shaped form embryos distinct from the species exposed to BIPP 11 (Figure
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2A and Figure 3g,h, Supplementary Table S3). Similar effects of TGF/TGFR inhibitors including
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SB431542 and RepSox on the sea urchin embryos from different species Strongylocentrotus purpuratus
and Lytechinus variegatus have been reported.31 PD173074, a potent inhibitor of both fibroblast growth
factor receptor 1 (FGFR1) and vascular endothelial growth factor receptor 2 (VEGFR2) inhibited spicule
growth at 1–4 M concentration. However, the agent did not cause spicule elimination contrasting the
observed BIPP effects (Supplementary Table S3). In addition, it was reported that the suppression of
FGFR1 signaling by specific antisense morpholino oligonucleotides in the sea urchin embryos induced
inhibition of skeletogenesis, gastrulation delay, prevented gut differentiation and pigment cells formation,32
the effects not observed for BIPP. Likewise, perturbations in the VEGF/VEGFR signaling network led to
the noticeable skeletal abnormalities along with the alteration of PMC migration.33, 34
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Similarly to the cytotoxicity assessment on the wild type and Notch-mutant leukemia cell lines, the
sea urchin embryo tests also confirmed Notch-independent mechanism of action of BIPP. Highly potent
and selective Notch/-secretase inhibitors semagacestat and LY-411575 along with the Notch signaling
inhibitor LY3039478 did alter spicule growth. However, as opposed to BIPP, they also induced a distinct
inhibition of archenteron differentiation, secondary mesenchyme cells, coelomic pouches and pigment
cells development (Figure 3i,j, Supplementary Table S3).
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Figure 3. Developmental alterations of the sea urchin embryos in response to different inhibitors of
spiculogenesis. (a) Intact mesenchyme blastula with primary mesenchyme cells (PMC) inside blastocoel.
(b) Hatching arrest caused by matrix metalloproteinase inhibitor marimastat (2 M). Mesenchyme blastula
developed inside fertilization envelope. (c) PMC elimination after exposure of zygotes to MEK1/2
inhibitor trametinib (20 nM). (d) Control plutei with skeletal spicules and differentiated gut. (e) MEK1/2
inhibitor binimetinib (500 nM) produced embryos without spicules and caused exogastrulation (outward
archenteron growth) with gut differentiation (f). (g) and (h) Bell-shaped radialized embryos exposed to
TGFR/Alk5 inhibitor RepSox at 0.2 M (g) and 1 M (h). (i) Suppression of archenteron (primary gut)
and secondary mesenchyme cells formation by Notch inhibitor LY3039478 (1 M). Some embryos
exogastrulated (j). Short irregular spicules are present. (a)–(c) 12 h postfertilization. Average embryo
diameter: 115 m. (e)–(j): 32–34 h postfertilization. Bar: 100 m. Incubation temperature: 24 °C.
Next, we probed other molecules for the selective cytotoxicity against MOLT-4 cells to identify
murrayafoline A as a candidate. This natural product isolated from the root bark of Murraya euchrestifolia
(Rutaceae) demonstrated potent selective cytotoxicity against MOLT-4 cells in NCI60 screen35 with
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MOLT-4 GI50 = 0.025 M (Supplementary Figure S24). It was found that murrayafoline A inhibited
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Wnt/-catenin signaling by -catenin degradation independent of GSK-3, -catenin phosphorylation and
Siah-1/APC pathway.36 This prompted us to test a hypothesis of potential targeting Wnt/-catenin
signaling cascade with BIPP. Specifically, we tested murrayafoline A as well as additional Wnt-
modulating agents (IWR-1-endo, XAV-939, IWP-2, LGK-974, Wnt-C59, NCB-0846, and BML-284) in
the sea urchin embryo model. The results are presented in Supplementary Table S3. Murrayafoline A
caused cleavage alteration (EC = ca 1 M) and cleavage arrest (EC = ca 8 M), when applied to fertilized
eggs. The arrested eggs acquired tuberculate shape typical of the effect caused by the microtubule
destabilizing agents. The same outcome was observed for a very potent and specific Wnt/PORCN
inhibitor, LGK-974, (EC = 1 and 4 M for cleavage alteration and cleavage arrest, respectively). Whereas
murrayafoline A and Wnt-targeting compounds inhibited spicule formation/growth, their phenotypic effect
was different compared to that of BIPP (Supplementary Table S3), specifically, neither spiculogenesis nor
hatching were affected significantly. Compound NCB-0846 was toxic resulting in the embryo mortality at
concentrations ≥0.2 M. Interestingly, known Wnt activator BML-284 caused cleavage alteration,
cleavage arrest and embryo spinning at 0.01, 0.05, and 0.2 M, respectively, evidencing strong antimitotic
microtubule destabilizing activity reported earlier using tubulin polymerization assay.37
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Considering the above, our results suggest that the molecular target(s) of BIPP 11 are unlikely to
involve MEK1/2, TGF/TGFR, FGFR, VEGF/VEGFR, Notch, and canonical Wnt/-catenin signaling
cascades. Furthermore, several additional molecular networks could be excluded from our evaluation. For
example, whereas inhibition of phosphoinositide 3-kinase (PI3K) altered spicule growth, it also blocked
embryo development at a gastrula stage.38 Acetazolamide, an inhibitor of carbonic anhydrase that regulates
biomineralization, suppressed spicule initiation and growth but also impeded PMC migration, prevented
pigment cells formation and caused general developmental delay.39 We did not observe these phenotypic
changes with BIPP 11 series. To further confirm the results of our studies, an investigation of NCI cancer
cell screen database (https://dtp.cancer.gov) for inhibitors of different signaling pathways did not reveal
any MOLT-4 selective compounds. Specifically, as suggested by NCI screen database, inhibitors of
MEK1/2 (cobimetinib, dabrafenib, sorafenib, and trametinib), VEGFR and FGFR (axitinib, cabozantinib,
CH5183284, dovitinib, pazopanib, ponatinib, regorafenib, and sorafenib), epidermal growth factor receptor
(EGFR) (afatinib, erlotinib, and lapatinib), PI3K (idelalisib, LY294002, and wortmannin), and Wnt/β-
catenin signaling (berberine, calotropin, chromomycin A2, nonactin, pyrvinium pamoate, and salinomycin)
did not exhibit any selective cytotoxicity against MOLT-4 cells.
CONCLUSIONS
In summary, 1,3-substituted pyrazolopyridinones 11–18 were synthesized using three-component domino
reaction, and their biological effects were evaluated on the sea urchin embryo model. BIPP 11 caused a
specific effect on the sea urchin embryos, namely inhibition of the embryo hatching and spiculogenesis.
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These compounds exhibited unique selectivity against MOLT-4 cancer cell line in the human cancer cell
screen. The sea urchin embryo test results indicated that BIPP cytotoxicity against MOLT-4 cells was
unrelated to the antimitotic tubulin machinery. Detailed SAR examination of these compounds suggested
that the observed MOLT-4 effect is likely a result of their interaction with the same cellular target(s)
and/or molecular networks in both assay systems. This assumption is supported by the fact that numerous
regulatory networks and signalling pathways common for other animals and humans were first described
and investigated comprehensively in the sea urchin embryos.40-43 Importantly, sea urchin and human
genomes share more than 7,000 genes.44 Subsequent studies of possible mechanisms that could mediate
MOLT-4 specificity allowed us to eliminate Notch, Wnt/-catenin, receptor tyrosine kinases
(VEGF/VEGFR, FGF/FGFR), PI3K, and Raf-MEK-ERK signaling pathways as possible targets of BIPP.
Although MMP-9/hatching enzyme was identified as one of the possible BIPP 11 targets, specific
cytotoxicity against MOLT-4 cells was unrelated to this mode of action.
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EXPERIMENTAL PROCEDURES
Chemistry. General Experimental Procedures. Melting points were measured on a Boetius melting
point apparatus and were uncorrected. 1H NMR and 13C NMR spectra were recorded on a Bruker DRX-
500 instrument [working frequencies of 500.13 MHz (1H) and 125.76 MHz (13C)]. Chemical shifts were
stated in parts per million (ppm) and referenced to the appropriate NMR solvent peaks. Spin-spin coupling
constants (J) were reported in Hertz (Hz). NMR spectra (Supporting Information) were prepared using an
original software designed at N. D. Zelinsky Institute of Organic Chemistry RAS (Moscow, Russian
Federation) (http://nmr.ioc.ac.ru:8080/SDF2PDF.kl1). Low resolution mass spectra (m/z) were recorded on
a Finnigan MAT/INCOS 50 mass spectrometer at 70 eV using direct probe injection. Elemental analysis
was performed on the automated PerkinElmer 2400 CHN microanalyzer. Flash chromatography was
carried out on silica gel (Acros, 0.035–0.070 mm, 60 Å). TLC was performed on Merck 60 F254 plates.
Solvents, Meldrum’s acid, 3-iminobutanenitrile and benzaldehydes 2a–k were purchased from Acros
Organics (Belgium) at the highest commercial quality and used as received. Non-commercial
benzaldehydes 2l–o45 and 2s14 were synthesized according to published procedures.
Synthesis of 1-(1H-Benzimidazol-2-yl)-3-methyl-1H-pyrazol-5-amine (3). A mixture of 2-
hydrazinyl-1H-benzimidazole (21) (15.67g, 0.106 M) and 3-oxobutanenitrile (8.8 g, 0.106 M) in EtOH
(100 mL) was heated for 40 min at 60–75 °C. The first precipitate was formed in 10 min. The reaction
mixture was cooled (3 °C), the precipitate was filtered off, washed with cold EtOH (10 mL), ether (10 mL),
and dried in air to afford 3 (12 g, 53% yield); mp 208–210 °C; 1H NMR (DMSO-d6, 500 MHz): δ 2.16 (s,
3H, CH3), 5.30 (s, 1H, H-4), 6.82 (s, 2H, NH2), 7.15 (m, 2H, H-5',6'), 7.39 (m, 1H, H-4’), 7.53 (m, 1H, H-
7'), 12.64 (s, 1H, NH); 13C NMR (DMSO-d6, 125 MHz) δ 13.8, 87.7, 111.0, 117.5, 121.6, 121.7, 132.3,
141.6, 147.5, 149.5, 151.6. EIMS m/z 213 [M+] (100), 173 (26), 133 (21), 132 (19), 118 (27), 105 (15).
Anal. Calcd for C11H11N5: C, 61.96; H, 5.20; N, 32.84. Found: C, 61.83; H, 5.17; N, 32.92;
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General Procedure for the Three Component Condensation. A mixture of 0.55 g (0.0038 mol)
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Meldrum’s acid 1 in 7 mL of AcOH, aldehyde 2 (0.0033 mol), and aminopyrazole 3–10 (0.003 mol) was
refluxed for 2 h. Then reaction mixture was concentrated in vacuo and the residue was triturated with 10
mL of EtOH. The resulting precipitate was filtered, washed with EtOH (3 × 7 mL) and dried to afford pure
compounds 11–18.
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General Procedure for Alkylation of 1-(Benzimidazol-2-yl)pyrazolo[3,4-b]pyridin-6-ones 11b and
11j. A mixture of 1-(benzimidazol-2-yl)pyrazolo[3,4-b]pyridin-6-one 11b or 11j (1 mmol), alkylating
agent (5 mmol MeI or 1.1 mmol n-C7H15I) and dried K2CO3 (1.1 mmol) in 3 mL of dry DMF was stirred at
room temperature for 3 h and then diluted with water (10 mL). To prepare the respective N-methyl
derivatives the resulting precipitate was filtered off, washed with water (2 mL) and EtOAc (2 mL) and
dried. In order to access N-heptyl derivatives, the resulting suspension was extracted with EtOAc (2 × 5
mL), the combined extracts were evaporated to dryness and the residue was purified by column
chromatography (heptane/EtOAc, 2:1). To synthesize the diaziridine derivative 11jz, a mixture of 11j (38
mg, 0.09 mmol), 3-(but-3-yn-1-yl)-3-(2-iodoethyl)-3H-diazirene (21 mg, 0.09 mmol) and dried К2СО3 (14
mg, 0.1 mmol) in 1 mL of dry DMF was stirred at 40–45 C for 6 h and diluted with water (5 mL). The
resulting suspension was extracted with EtOAc (2 × 5 mL), the combined extracts were concentrated in
vacuo and the residue was purified by column chromatography (heptane/EtOAc, 2:1).
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Biology. Materials and Methods. AS703026, AZ628, AZD8330, BML-284, cobimetinib, CTS-
1027, dabrafenib, FR 180204, GDC-0623, GDC-0879, GDC-0994, GM6001, ITD-1, IWP-2, LY3039478,
LY3200882, LY-411575, marimastat, NCB-0846, PD184352, PD318088, refametinib, RepSox,
RO4987655, SB590885, SCH772984, selumetinib, semagacestat, T-5224, TAK-632, TAK-733,
trametinib, ulixertinib, VX-11e, and Wnt-C59 were purchased from MedChem Express LLC, Monmouth
Junction, NJ, USA. Binimetinib, EW-7197, GW788388, IWR-1-endo, LGK-974, PD173074, SB431542,
and XAV-939 were purchased from Selleckchem, Houston, TX, USA. Murrayafoline A was purchased
from Ark Pharm Inc., Arlington Heights, IL, USA. CGS 27023A was provided by Novartis. 3-(But-3-yn-
1-yl)-3-(2-iodoethyl)-3H-diazirine was prepared as described previously.18
Resazurin (Alamar Blue) Cytotoxicity Assay.15 The leukemia cell lines were obtained from the
American Type Culture Collection (ATCC; HL-60, LOUCY, Mino, MOLT-4, MOLT-16, and Rec-1) and
German Collection of Microorganisms and Cell Cultures GmbH (DSMZ; HPB-ALL, Jeco-1, Jurkat,
RPMI-8402, and SUP-T11) and maintained in RPMI-1640 supplemented with 10–20% fetal bovine serum
(Amimed #2-01F30-I)) and 2 mM L-glutamine (Supplementary Table S4). Cell lines were seeded at
40.000 cells per well in 100 μL of complete media in 96-well tissue-culture-treated microplates, flat
bottom (TPP#92696). Compounds (10 mM in DMSO) were transferred to cells seeded in 96-well plates
using HP dispenser to create a ten-point dose–response curve in triplicate starting from 1 uM with a 1:3
serial dilution. Cells were cultured in a humidified incubator set at 37 °C and maintained at an atmosphere
of 5% CO2. On Day 4, 10 μL per well of the resazurin reagent (Sigma #R7017 diluted in PBS as a stock
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solution at 130 μg/mL, stored at 4 °C) was added. The plates were incubated at 37 °C for 4 h in an
incubator after which fluorescence was read using a fluorescence plate reader at 560ex/590em, sensitivity
40 (Synergy HT from Biotek and Gen5 software). Normalized fluorescence intensity data were analyzed
using XLfit, and subsequent IC50 values were calculated using Dose Response one site 4 parameter logistic
model.
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Biochemical Screen on Proteases. All protein and peptide containing solutions were handled in
siliconized tubes (Life Systems Design, Merenschwand, Switzerland). The compound solutions were
transferred to 384-well assay plates (black Cliniplate; cat. no. 95040020 Labsystems Oy, Finland) by
means of a CyBi-Hummingwell pipettor (CyBio AG, Jena, Germany). The enzyme and substrate solutions
were transferred to the assay plates by means of a CyBi-well 384-channel pipettor (CyBio AG, Jena,
Germany). For the determination of IC50 values, the assays were performed at room temperature in 384-
well plates with a total assay volume of 25.25 L per well. The test compounds were dissolved in 90 %
(v/v) DMSO/water. For the assays, 250 nL of the 90 % (v/v) DMSO/water solution or compound solution
were added per well, followed by the addition of 12.5 L protease solution (protease in buffer solution).
The final assay concentrations of each enzyme are given in section Assay conditions. After 70 min of pre-
incubation at room temperature, the reactions were started by the addition of 12.5 L substrate solution
(see section Assay conditions). After the addition of the substrate solution, the final DMSO concentration
in the assay was 0.9 % (v/v). The effect of the compound on the enzymatic activity was obtained from the
linear part of the progress curves and determined after 1 h (t = 60 min). The final compound concentrations
ranged from 100 µM to 1 nM. Plate measurements were conducted with an Ultra reader (TECAN,
Maennedorf, Switzerland). For fluorescence intensity (FI) measurements with the dye pairs
EDANS/DABCYL and Mca/Dnp or the dye AMC, the instrument was equipped with a combination of a
350 nm (20 nm bandwidth) and a 500 nm (25 nm bandwidth) bandpass filter for fluorescence excitation
and emission acquisition, respectively. For FI measurements with the dye Rh110, the instrument was
equipped with a combination of a 485 nm (25 nm bandwidth) and a 535 nm (25 nm bandwidth) bandpass
filter for fluorescence excitation and emission acquisition, respectively. To increase the signal:background
ratio, appropriate dichroic mirrors were employed. All filters and dichroic mirrors were purchased from
TECAN. The fluorophores in each well were excited by three flashes per measurement. The IC50 values
were calculated from the plot of percentage of inhibition vs. inhibitor concentration by a logistics fit
according to
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y = A2 + (A1 – A2) / (1 + (x/ IC50) ^ p)
(Equation 1)
where y is the %-inhibition at the inhibitor concentration, x. A1 is the lowest inhibition value, i.e. 0 %, and
A2 the maximum inhibition value, i.e. 100 %. The exponent, p, is the Hill coefficient. The curve fitting
was conducted with the non-linear regression routine of the in-house software Helios.
Enzymes:
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human MMP1 (Uniprot P03956) covering amino acids 1-469; expressed in and purified from stable
transfected C127 cells
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human MMP2 (Uniprot P08253) covering amino acids 1-660; expressed in and purified from insect cells
(baculovirus expression system)
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human MMP7 (Uniprot P09237) covering amino acids 95-267; expressed in and purified from E.coli
human MMP8 (Uniprot P22894) covering amino acids 100-262; expressed in and purified from E.coli
human MMP9 (Uniprot P14780) covering amino acids 1-707; expressed in and purified from stable
transfected 293 cells
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human MMP12 (Uniprot P39900) covering amino acids 101-268; expressed in and purified from E.coli
human MMP13 (Uniprot P45452) covering amino acids 103-274; expressed in and purified from E.coli
human MMP14 (Uniprot P50281) covering amino acids 112-284; expressed in and purified from E.coli
human TACE (Uniprot P78536) covering amino acids 1-670; expressed in and purified from insect cells
(baculovirus expression system)
substrate: MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2, purchased from Biosyntan (www.biosyntan.de),
product number 6375
enzyme concentrations: 0.3 nM (MMP1), 8 nM (MMP2), 0.4 nM (MMP7), 1 nM (MMP8), 0.3 nM
(MMP9), 1.5 nM (MMP12), 0.015 nM (MMP13), 0.2 nM (MMP14), 0.3 nM (TACE)
substrate concentration: 5 mM
assay buffer: 100 mM Tris, pH 7.4, 100 mM NaCl, 10 mM CaCl2, 10 mM ZnCl2, 0.075% (v/v) Brij35
Phenotypic Sea Urchin Embryo Assay.13, 14 Adult sea urchins, Paracentrotus lividus (Echinidae),
were collected from the Mediterranean Sea on the Cyprus coast and kept in an aerated seawater tank.
Gametes were obtained by intracoelomic injection of 0.5 M KCl. Eggs were washed with filtered seawater
and fertilized by adding drops of diluted sperm. Embryos were cultured at room temperature under gentle
agitation with a motor-driven plastic paddle (60 rpm) in filtered seawater. The embryos were observed
with a Biolam light microscope (LOMO, St. Petersburg, Russia). Microphotographs were obtained using
an AmScope binocular microscope with an MU500 digital camera (United Scopes LLC, Irvine, CA, USA).
For treatment with the test compounds, 5 mL aliquots of embryo suspension were transferred to six-well
plates and incubated as a monolayer at a concentration up to 2000 embryos/mL. Stock solutions of tested
molecules were prepared in DMSO at 10 mM concentration followed by a 10-fold dilution with 96%
EtOH. This procedure enhanced the solubility of the test compounds in the salt-containing medium
(seawater), as evidenced by microscopic examination of the samples. The maximal tolerated
concentrations of DMSO and EtOH in the in vivo assay were determined to be 0.2% and 0.5%,
respectively. Higher concentrations of either DMSO (0.5%) or EtOH (1%) caused nonspecific alteration
and retardation of the sea urchin embryo development independent of the treatment stage. The
antiproliferative activity was assessed by exposing fertilized eggs (8–15 min after fertilization, 45–55 min
before the first mitotic cycle completion) to 2-fold decreasing concentrations of the compound. Cleavage
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alteration and arrest were clearly detected at 2.5 h and 5.5 h after fertilization, when control embryos
reached 8-cell and early blastula stages, respectively. The effects were estimated quantitatively as an
effective threshold concentration, resulting in cleavage alteration and embryo death before hatching or full
mitotic arrest. At these concentrations all tested microtubule destabilizers caused 100% cleavage alteration
and embryo death before hatching, whereas at 2-fold lower concentrations the compounds failed to
produce any effect. For microtubule-destabilizing activity, the compounds were tested on free-swimming
blastulae just after hatching (8–10 h postfertilization), which originated from the same embryo culture.
Embryo spinning was observed after 15 min to 20 h of treatment, depending on the structure and
concentration of the compound. Both spinning and lack of forward movement were interpreted to be the
result of the microtubule-destabilizing activity of a molecule. Video illustrations are available at
http://www.chemblock.com. Sea urchin embryo assay data are available at http://www.zelinsky.ru.
Experiments with the sea urchin embryos fulfill the requirements of biological ethics. The artificial
spawning does not cause animal death, embryos develop outside the female organism, and both post
spawned adult sea urchins and the excess of intact embryos are returned to the sea, their natural habitat.
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ASSOCIATED CONTENT
Supporting Information
Analytical data for compounds 11–18; NCI60 screen graphs for compounds 11a, 11b, 11f, 11g, 11i, 11j,
11k, 11q, 11r, 16o, and murrayafoline A; kinase screen and RF-QSAR data for compound 11j; effects of
inhibitors of spiculogenesis; leukemia cell lines description; schematic diagram of skeletogenic primary
mesenchyme cells differentiation (Tables S1–S4, Figures S1–S24); 1H NMR spectra of compounds 11–18
(PDF)
AUTHOR INFORMATION
Corresponding Author
*Tel: +7 916 620 9584. Fax: +7 499 137 2966. E-mail: vs@zelinsky.ru.
ORCID
Victor V. Semenov: 0000-0003-1889-341X
Alexei S. Karpov: 0000-0002-3067-4538
Alexander V. Samet: 0000-0002-0100-085X
Marina N. Semenova: 0000-0001-7656-9023
Author Contributions
B.L., A.Kar., M.S., A.Kis. and V.S. conceived and designed the experiments, A.K., A.D., M.K., E.Kh.,
A.S., E.S., D.G., T.R., M.S. performed the experiments, A.Kar., L.K., M.S., A.Kis. and V.S. co-wrote the
manuscript and Supporting Information. All authors have given approval to the final version of the
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manuscript. We also wish to thank Dr. Yu. A. Strelenko from N. D. Zelinsky Institute of Organic
Chemistry RAS for the development of NMR spectra presentation software.
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Funding
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The Russian Science Foundation, Grant 18-13-00044.
Notes
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank the National Cancer Institute (NCI) (Bethesda, MD, USA) for screening compounds by the
Developmental Therapeutics Program at NCI (Anticancer Screening Program; http://dtp.cancer.gov). We
wish to thank Academics Relations and Operational Alliances at Novartis for the general support. We are
grateful to U. Hassiepen and Q. Zhiyan for their excellent technical assistance.
Abbreviations:
BIPP, benzimidazolyl-pyrazolo[3,4-b]pyridinone
EGFR, epidermal growth factor receptor
ERK, extracellular signal-regulated kinase
FGF/FGFR, fibroblast growth factor/fibroblast growth factor receptor
MAPK, mitogen-activated protein kinase
MMP, matrix metalloproteinase
PI3K, phosphoinositide 3-kinase
PMC, primary mesenchyme cells
SAR, structure-activity relationship
SRB, sulforhodamine B
T-ALL, T-cell acute lymphoblastic leukemia
TGF/TGFR, transforming growth factor /transforming growth factor  receptor
VEGF/VEGFR, vascular endothelial growth factor/vascular endothelial growth factor receptor
Table of Content Graphic
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