Sea Urchin Embryo Model As a Reliable in Vivo Phenotypic Screen to Characterize Selective Antimitotic Molecules. Comparative evaluation of Combretapyrazoles, -isoxazoles, -1,2,3-triazoles, and -pyrroles as Tubulin-Binding Agents

Article abstract of DOI:10.1021/acscombsci.8b00113

A series of both novel and reported combretastatin analogues, including diarylpyrazoles, -isoxazoles, -1,2,3-triazoles, and -pyrroles, were synthesized via improved protocols to evaluate their antimitotic antitubulin activity using in vivo sea urchin embryo assay and a panel of human cancer cells. A systematic comparative structure-activity relationship studies of these compounds were conducted. Pyrazoles 1i and 1p, isoxazole 3a, and triazole 7b were found to be the most potent antimitotics across all tested compounds causing cleavage alteration of the sea urchin embryo at 1, 0.25, 1, and 0.5 nM, respectively. These agents exhibited comparable cytotoxicity against human cancer cells. Structure-activity relationship studies revealed that compounds substituted with 3,4,5-trimethoxyphenyl ring A and 4-methoxyphenyl ring B displayed the highest activity. 3-Hydroxy group in the ring B was essential for the antiproliferative activity in the diarylisoxazole series, whereas it was not required for potency of diarylpyrazoles. Isoxazoles 3 with 3,4,5-trimethoxy-substituted ring A and 3-hydroxy-4-methoxy-substituted ring B were more active than the respective pyrazoles 1. Of the azoles substituted with the same set of other aryl pharmacophores, diarylpyrazoles 1, 4,5-diarylisoxazoles 3, and 4,5-diaryl-1,2,3-triazoles 7 displayed similar strongest antimitotic antitubulin effect followed by 3,4-diarylisoxazoles 5, 1,5-diaryl-1,2,3-triazoles 8, and pyrroles 10 that showed the lowest activity. Introduction of the amino group into the heterocyclic core decreased the antimitotic antitubulin effect of pyrazoles, triazoles, and to a lesser degree of 4,5-diarylisoxazoles, whereas potency of the respective 3,4-diarylisoxazoles was increased.

Full text of DOI:10.1021/acscombsci.8b00113

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Article
The sea urchin embryo model as a reliable in vivo phenotypic
screen to characterize selective antimitotic molecules.
Comparative evaluation of combretapyrazoles, -isoxazoles,
-1,2,3-triazoles, and -pyrroles as tubulin binding agents.
Marina N. Semenova, Dmitry V. Demchuk, Dmitry V. Tsyganov, Natalia B. Chernysheva, Alexander
V. Samet, Eugenia A. Silyanova, Victor P. Kislyi, Anna S. Maksimenko, Alexander E. Varakutin,
Leonid D. Konyushkin, Mikhail M. Raihstat, Alex S. Kiselyov, and Victor Vladimirovich Semenov
ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.8b00113 • Publication Date (Web): 19 Nov 2018
Downloaded from http://pubs.acs.org on November 19, 2018
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The sea urchin embryo model as a reliable in vivo phenotypic screen to characterize
selective antimitotic molecules. Comparative evaluation of combretapyrazoles, -isoxazoles,
-1,2,3-triazoles, and -pyrroles as tubulin binding agents
†
‡
‡
‡
Marina N. Semenova, Dmitry V. Demchuk, Dmitry V. Tsyganov, Natalia B. Chernysheva,
‡
‡
‡
‡
Alexander V. Samet, Eugenia A. Silyanova, Victor P. Kislyi, Anna S. Maksimenko,
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Alexander E. Varakutin, Leonid D. Konyushkin, Mikhail M. Raihstat, Alex S. Kiselyov,
Victor V. Semenov,*,‡
†
N. K. Koltzov Institute of Developmental Biology RAS, 26 Vavilov Street, 119334 Moscow,
Russian Federation
‡
N. D. Zelinsky Institute of Organic Chemistry RAS, 47 Leninsky Prospect, 119991 Moscow,
Russian Federation
§
Genea Biocells US Inc., Suite 210, 11099 North Torrey Pines Rd., La Jolla, CA 92037, USA
Corresponding author: Victor V. Semenov
Address: N. D. Zelinsky Institute of Organic Chemistry, RAS, Leninsky Prospect, 47, 119991,
Moscow, Russian Federation. Tel.: +7 916 620 9584; fax: +7 499 137 2966.
E-mail: vs@zelinsky.ru
E-mail addresses:
Marina N. Semenova
Dmitry V. Demchuk
Dmitry V. Tsyganov
Natalia B. Chernysheva
Alexander V. Samet
Eugenia A. Silyanova
Victor P. Kislyi
ms@chemical-block.com
demchuk@ioc.ac.ru
tsyg@ioc.ac.ru
info@chemblock.com
sametav@ioc.ac.ru
cea93@mail.ru
vkislyi@yandex.ru
Anna S. Maksimenko
Alexander E. Varakutin
Leonid D. Konyushkin
Mikhail M. Raihstat
Alex S. Kiselyov
chem.annamaks@gmail.com
waralex1996@gmail.com
LeonidK@chemical-block.com
mr@chemical-block.com
alex.kiselyov@geneabiocells.com
vs@zelinsky.ru
Victor V. Semenov
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ABSTRACT
A series of both novel and reported combretastatin analogues including diarylpyrazoles, -
isoxazoles, -1,2,3-triazoles and -pyrroles were synthesized via improved protocols in order to
evaluate their antimitotic antitubulin activity using in vivo sea urchin embryo assay and a panel
of human cancer cells. A systematic comparative structure-activity relationship studies of these
compounds were conducted. Pyrazoles 1i and 1p, isoxazole 3a, and triazole 7b were found to be
the most potent antimitotics across all tested compounds causing cleavage alteration of the sea
urchin embryo at 1 nM, 0.25 nM, 1 nM, and 0.5 nM, respectively. These agents exhibited
comparable cytotoxicity against human cancer cells. Structure-activity relationship studies
revealed that compounds substituted with 3,4,5-trimethoxyphenyl ring A and 4-methoxyphenyl
ring B displayed the highest activity. 3-Hydroxy group in the ring B was essential for the
antiproliferative activity in the diarylisoxazole series, whereas it was not required for potency of
diarylpyrazoles. Isoxazoles 3 with 3,4,5-trimethoxy substituted ring A and 3-hydroxy-4-methoxy
substituted ring B were more active than the respective pyrazoles 1. Of the azoles substituted
with the same set of other aryl pharmacophores, diarylpyrazoles 1, 4,5-diarylisoxazoles 3, and
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,5-diaryl-1,2,3-triazoles 7 displayed similar strongest antimitotic antitubulin effect followed by
,4-diarylisoxazoles 5, 1,5-diaryl-1,2,3-triazoles 8, and pyrroles 10 that showed the lowest
activity. Introduction of the amino group into the heterocyclic core decreased the antimitotic
antitubulin effect of pyrazoles, triazoles, and to a lesser degree of 4,5-diarylisoxazoles, whereas
potency of the respective 3,4-diarylisoxazoles was increased.
Keywords:
Combretastatins
Diarylazoles
Sea urchin embryo
Antimitotic
Microtubule destabilization
Abbreviations:
CA2, combretastatin A-2
CA4, combretastatin A-4
SAR, structure-activity relationship
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INTRODUCTION
Cytostatic agents that affect structure and/or dynamics of microtubules attract
considerable attention in the development of novel antitumor agents that are potent, selective,
address cancer resistance, and display more favorable therapeutic window. An appropriate
integration of target-based and phenotypic screening is a very promising approach in drug
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discovery. In this regard a choice of proper model is of a paramount importance in the
phenotypic screening. Ideally, a model organism should be readily available, accessible, and
feasible. It should also furnish data relevant to human physiology. While searching for novel
microtubule dynamics modulating compounds, we selected a sea urchin embryo model for the
phenotypic assay. This organism is well characterized and widely used in experimental biology
to study various aspects of embryology, developmental and molecular biology, genetics and
ecology.2–8 There are multiple advantages to a sea urchin embryo over alternative biological
models. These include a) simple access to a large number of gametes, b) facile artificial
fertilization and rearing, c) quick and synchronous cell division during early embryogenesis, d)
well studied differentiation and morphogenesis, e) direct relevance to mammalian and human
signaling networks. Relatively large size and optical transparency of sea urchin eggs, embryos
and larvae allows for convenient monitoring of the assay with conventional microscope. Sea
urchin embryos can be reliably reared in filtered seawater without need for specialized
equipment, culture media, or sterile conditions. Sea urchin and human genomes share more than
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,000 genes including orthologs associated with a number of human disorders.9, 10 From the
evolutionary point of view, sea urchins are more related to humans than other model organisms
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including fruit fly Drosophila melanogaster and nematode Caenorhabditis elegans. A number
of fundamental aspects of embryogenesis, regulatory pathways and signalling macromolecules,
which play key roles in all animal and human cells, were first discovered in the sea urchin
embryos.2–4 As a result, these represent a robust and versatile model organism for developmental
biology and biomedical research.10–13 Furthermore, experimental settings using the sea urchin
embryos are ethical since the artificial spawning does not result in animal death, embryos
develop outside the female organism, and both post spawned adult sea urchins and the excess of
intact embryos can be released back to the sea, their natural habitat.
Several research teams have been using sea urchin embryos to identify small molecules
that modulate cell division.14–20 Following both literature evidence as well as our internal
experience with the organism, we applied the sea urchin embryos to identification of antimitotics
with microtubule destabilizing mode of action. This decision was based on the unique
developmental features of sea urchin embryos that rely on microtubule dynamics, namely, two
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distinct phases of embryogenesis that can be easily observed in situ. These phases include a)
microtubule-dependent blastomere divisions during cleavage stage that occur every 35–40 min
Figure 1, A) and b) active swimming of hatched embryos that is mediated by the coordinated
(
beating of numerous cilia comprised of microtubules. We reasoned that an antitubulin agent
would affect both cell division and embryo motility thus making the sea urchin embryo to be a
convenient phenotypic model. Notably, in our hands treatment of the embryos with diverse
microtubule destabilizing agents including colchicine, nocodazole, podophyllotoxin,
combretastatins, vinblastin, and dolastatin 15 selectively affect cleavage and alter swimming
behavior. While intact embryos move rapidly at the surface of the seawater with animal pole
forward, slowly rotating around animal-vegetal axis, microtubule destabilizers cause the
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organism to stop swimming forward, settle on the bottom of the vessel, and spin. Intriguingly,
while a microtubule stabilizer paclitaxel (Figure 1, G) along with several non-tubulin acting
antimitotics do alter cellular division, these agents fail to induce spinning of the hatched
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embryos. In addition, eggs arrested by microtubule destabilizers acquire specific tuberculate
shape (Figure 1, E), which could also be considered as an indirect indication of their antitubulin
effect. Combination of these phenotypic features resulted in development of simple, reliable, and
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efficient in vivo phenotypic assay that identifies microtubule-specific antimitotics. Importantly,
the sea urchin embryo assay combines both phenotypic and target-based screening as it shows
test molecule effect on the dividing cells of the developing organism (Figure 1) and provides
evidence for the microtubule destabilizing mode of action. The protocol was further validated
using conventional purified tubulin polymerization and cell-based assays22–24 to show good
correlation of data for the reported actives. It should be noted that the sea urchin embryo system
also allows for the identification of compounds that display non-tubulin antiproliferative activity,
selective effects on distinct morphogenetic events, and systemic toxicity.
Next, we established the optimized rearing conditions for the assay. Specifically, the best
concentration of the sea urchin early embryos was found to be in the 400–2500 eggs
(embryos)/mL interval to provide an even monolayer suitable for reproducible and tractable
screening. Increasing embryo count could result in nonselective absorbtion of the tested
molecule by the embryo. Moreover, 6-well plates offered favorable topology/volume (5 mL).
Large diameter and small well depth were of particular importance to secure normal
development of the sea urchin embryos, sufficient aeration and monolayer in particular. In
contrast, test runs using 24-well plates and 1 mL well volume were unsuccessful primarily due to
the observed embryo malformations. It was a combination of both specific culture conditions and
lack of an automation to track specific changes in the embryo’s motility (spinning instead of
forward swimming) that limited our ability to improve the assay throughput. Despite of these
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challenges, the sea urchin embryo model offers considerable advantages in the phenotypic
screening. These include: i) facile and rapid access to a large number of gametes and eggs
immediately suitable for screening; ii) efficient incubation with no need for chemicals and/or
specific media, sterility or specialized equipment; iii) feasible monitoring of the phenotypic
effects that could be accomplished via a conventional low magnification light microscope; iv)
sound reproducibility.
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Figure 1. Typical effects of tubulin/microtubule targeting compounds on the sea urchin
Paracentrotus lividus eggs and embryos. (A–C) Control. (A) Intact egg with normal bipolar
mitotic spindle (light spots), the first cleavage anaphase. (B) Eight-cell embryo. (C) Early
blastula. (D–F) Effects of the microtubule destabilizer combretastatin A-4 at 20 nM (D and E)
and 5 nM (F). (D) Arrested egg without mitotic apparatus. (E) Tuberculate arrested eggs. (F)
Cleavage alteration. (G) Aberrant multipolar mitotic apparatus in arrested egg in the presence of
the microtubule stabilizer paclitaxel (5 M). Compounds were added to zygotes at 8–15 min
postfertilization. Samples were incubated at 21 C. Eggs/embryos were observed at 1 h (A), 2.5 h
(B, D, E, G), and 6 h (C, F) postfertilization. The average egg/embryo diameter is 115 m.
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Considering potential for inadequate compound solubility that often hampers screening
procedures and could resulted in inconsistent data, we streamlined sample preparation by
diluting the initial 10 mM DMSO compound solutions by 10 fold with 96% EtOH. In our hands,
this step dramatically enhanced solubility of the test molecules in seawater, as evidenced by the
microscopy examination of samples. For the sea urchin embryos, maximal tolerated
concentrations of solvents and surfactants commonly used in medicinal chemistry were
determined as well (Table 1) (Semenova M.N., unpublished).
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Table 1. Toxicity of Selected Solvents and Surfactants on Sea Urchin Paracentrotus lividus
Embryos (% Concentration)
effects
developmental
delay/alteration
1
systemic toxicity/
solvent/surfactant
Ethanol
no effect
0.5
mortality
1.5
Dimethylsulfoxide
Dimethylformamide
Dimethylacetamide
0.2
0.5
>1
0.2
0.5
1
0.02
0.1
0.04–0.5
0.2–0.5
0.05–0.2
0.005–0.02
0.005-0.01
NAa
1
2
-Pyrrolidone
1
N-Methyl-pyrrolidone
Cremophor EL
Sorbitan monolaurate
PEG 300
0.02
0.002
0.002
0.2
0.5-1
>0.02
0.013
0.5-1
0.6
PEG 6000
0.12
0.3
a
NA: not available.
Isosteric replacement of the labile cis-double bond in a highly potent natural antimitotic
combretastatin A-4 (CA4, Figure 2) is a feasible strategy to access novel tubulin modulators with
enhanced physiochemical properties.25–29 The ethene bridge furnishes critical cis-configuration
of the biaryl template necessary for the optimized interaction of a molecule with the colchicine
binding site of tubulin.30, 31 Five-membered N-containing heterocycles were reported to provide a
non-isomerizable and metabolically stable isosteric replacement for the cis-styrene.25–29 Several
research groups including our team designed expeditious synthetic routes to respective five-
membered N-heterocycles termed combretazoles that recapitulate cis-biaryl topology for rings A
and B of the parent CA4 and display activity both in vitro and in vivo.32–55 However, the direct
comparison of compounds activities and/or general structure-activity relationship (SAR) across
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multiple classes of comretazoles are challenging primarily due to the formidable controversies
associated with their cytotoxicity and in vitro inhibition of purified tubulin polymerization, as
reported by multiple authors. For example, published ITP (concentration causing 50%
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inhibition of purified tubulin polymerization) values for the parent CA4 range between 0.175,56
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6.0, and 12.7 M. There are also considerable discrepancies in the reported GI values for
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human cancer cell lines (Table 2), which are likely the result of different experimental protocols,
improper identification and/or contamination of cancer cell lines.59
Table 2. Human Cancer Cell Growth Inhibition by CA4
GI , nM
5
0
NCI cancer
screen data
1–2.5
ref. 35
350
ref. 60
ref. 42
ref. 39
46
ref. 61
cell linea
K-562
10
HCT-116
HT29
1–3.2
100–1600
1
270
220
4200
1000
PC-3
5400
MCF7
5
a
K562: myelogenous leukemia; HCT-116: colon carcinoma; HT29: colon adenocarcinoma; PC-
3: prostate adenocarcinoma; MCF7: breast adenocarcinoma.
Moreover, inconsistent data persistent in the literature on the biological activity of ortho-
biaryl substituted pyrazoles, isoxazoles and triazoles also may have resulted from the inaccurate
determination of chemical structure, insufficient purity55, 62 or solubility of the tested molecules.
In our hands some azole derivatives of CA4 precipitated in the assay medium (seawater) upon
dilution of respective DMSO stocks to afford fine crystals visible under a microscope.
Considering these discrepancies, we decided to conduct a systematic head-to-head structure-
activity relationship (SAR) studies of the reported and novel active pyrazole, isoxazole, 1,2,3-
triazole-, and pyrrole-based analogues of CA4 and combretastatin A-2 (CA2) (Figure 2) using in
vivo phenotypic sea urchin embryo assay and in vitro cancer cell line cytotoxicity data. As a part
of the project, we synthesized CA4 and CA2 analogues that exhibit relevant substitution pattern
in the rings A and B as well as their derivatives with amino group in the N-heterocycle.
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Bridge
HN
N
O N
N O
RO
RO
R3
R3
R3
R1
R1
R1
A
R2
R2
R2
B
OCH3
OH
OCH3
Pyrazoles
: R = H
4,5-Diaryl-isoxazoles
3,4-Diaryl-isoxazoles
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3: R = H
5: R = H
3
3
3
CA2: R = -CH2-
CA4: R = CH3
2
: R = NH
4: R = NH
6: R = NH
3 2
3
2
3
2
N
N
N
N
NH
NH
N
R3
R1
R1
R1
R2
R2
R2
4
,5-Diaryl-1,2,3-triazoles
1,5-Diaryl-1,2,3-triazoles
Pyrroles
7
8: R = H
10
3
9: R = NH
3
2
Figure 2. Structures of combretastatins A-4 and A-2 and their cis-restricted azole analogues.
RESULTS AND DISCUSSION
Chemistry. While the respective direct analogues of CA4 including combretapyrazoles
5
3
33, 34
1a, 1c and combretaisoxazoles 3a, 3b,
(Schemes 1 and 2) have been described in the
literature, their synthesis was rather convoluted. In order to streamline the approach to relevant
combretapyrazoles 1a and 1b we introduced a robust synthetic protocol that included
iodosodiacetate-mediated rearrangement of easily accessible chalcones 13a,b into ketals 14a,b
followed by their in situ cyclization to furnish the targeted pyrazoles (Scheme 1).
Scheme 1. Synthesis of 4,5-Diarylpyrazoles 1 and 4,5-Diarylisoxazoles 3a
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O
OH
O
O
O
R
R
OCH3
R
OH
a
Isovanillin
H CO
H CO
H CO
OCH3
3
3
3
b
R
R
R
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1a,b
12a,b
13a,b 56-61%
c
OCH3
OH
O
O
O
O
R
R
d
H CO
H CO
OCH3
3
3
H CO
OCH3
R
3
R
1
4a,b in situ 70-80%
13-Ac a,b 72-81%
e
f
N
N
O
HN
R
H CO
3
a: R = OCH3
b: R = H
H CO
H CO
3
3
R
H CO
3
OH
OCH3
OH
OCH3
1
1
a: 72%
b: 63%
3
a 55%
a
Reagents and conditions: (a) ref. 63; (b) NaOH–EtOH, rt, 24 h; (c) AcCl–Pyridine, CH Cl , rt, 3
2
2
+
+
+
h; (d) PhI(OAc) , MeOH–H , rt, 8 h; (e) EtOH–H , N H 2HCl, reflux, 1 h; (f) EtOH–H ,
2
2
4
NH OHHCl, reflux, 2 h.
2
The corresponding combretapyrazole isomer 1c and respective analogues were obtained
via similar approach from guaiacol and respective benzaldehydes. Conveniently, the protective
acetate functionalities were successfully removed during the isomerization of 13 (Scheme 2).
Scheme 2. Synthesis of 4,5-Diarylpyrazoles 1 and 4,5-Diarylisoxazoles 3a
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
R1
O
R2
R3
O
O
R1
O
1
1
OH
O
R4
R2
R3
OH
a
b
OCH3
OCH3
OCH3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
12
R4
13
Guaiacol
c
R2
R3
R1
O
O
R1
O
OH
R2
R3
O
d
R4
OCH3
OCH3
H CO
OCH3
3
R4
14 in situ 84%
13-Ac 88%
e
f
R1
N
R1
N
O
NH
R2
R3
R2
R3
R4
R4
OH
OCH3
OH
OCH3
1c: R = H, R = R = R = OCH (12%)
3b: R = H, R = R = R = OCH (26%)
1 2 3 4 3
1
2
3
4
3
1
1
d: R = R = R = H, R = OCH (48%)
3c: R = H, R = OCH , R , R = -OCH O- (18%)
1 4 3 2 3 2
1
2
4
3
3
e: R = R = OCH R , R = -OCH O- (18%)
3d: R = R = OCH , R , R = -OCH O- (23%)
1 4 3 2 3 2
1
4
3,
2
3
2
3e: R = R = R = H, R = OCH (52%)
1
2
4
3
3
a
Reagents and conditions: (a) ref. 64; (b) NaOH–EtOH, rt, 24 h; (c) AcCl–Pyridine, CH Cl , rt, 3
2
2
+
+
+
h; (d) PhI(OAc) , MeOH–H , rt, 8 h; (e) EtOH–H , N H 2HCl, reflux, 1 h; (f) EtOH–H ,
2
2
4
NH OHHCl, reflux, 2 h.
2
Combretaisoxazole-based isomers 3a and 3b were synthesized following the same
synthetic route (Schemes 1 and 2). In general, the absence of –OH functionalities in the Ph-ring
facilitated access to the intermediate chalcones 13 (Scheme 3) that rearranged in situ to afford
ketoacetals 14 (path A) or an epoxy-intermediate 15 to yield ketoaldehydes 16 (path B).
Importantly, in many instances neither 14 nor 16 were isolated. Instead, the reaction mixture was
treated with NH NH or NH OH to result in pyrazoles 1 or isoxazoles 3.
2
2
2
Scheme 3. Synthesis of 4,5-Diarylpyrazoles 1a
1
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8
9
O
R1
O
O
O
R1
R4
O
Path B
e
R1
R5
R2
R5
R2
R5
R6
R2
R3
a
+
R6
R3
R6
R3
R7
R4
R7
R7
R4
1
5 65-89%
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
12
13 62-89%
Path A
b
f
R2
R3
R1
R1
N
O
O
R1
O
O
R2
R2
R5
c
g
d
R5
R4
R3
R3
R6
R6
H CO
OCH3
R4
3
R7
R5
R4
R7
R7
1
6
R6
1
4 67%
3h-j
d
N
R1
N
N
HN
H CO
R2
NH
H CO
3
NH
3
h
h
H CO
H CO
R3
3
3
H CO
H CO
R4
3
3
R7
R5
NH2
NH2
OCH3
R6
1i
OCH3
1
h
1
f,g,j-n
Path A, B
Path A
R1
R2
R3
R4
R5
R6
R7
Yield Path
1
f:
H
H
H
H
H
NO2
OCH3
H
OCH3 OCH3 OCH3 62%
A
1g:
OCH3
OCH3 OCH3 NO2
OCH3
H
23% AB
1
1
1
1
1
3
j:
OCH C H OCH3
H
H
OCH3 OCH3 OCH3 52%
A
A
B
A
B
A
A
A
A
B
2
6
5
k:
l:
OCH C H OCH3
H
OCH3
H
H
H
H
H
H
H
H
H
62%
17%
46%
61%
56%
54%
75%
70%
18%
2
6
5
OCH3
OCH3 OCH3 -OCH CH O-
2
2
m: OCH3
-OCH O-
OCH3
H
H
OCH3
2
n:
H
-NHCOCH O-
OCH3
OCH3
2
f: OCH3
-OCH O-
OCH3
OCH3 OCH3
2
3g: OCH3
h: OCH3
-OCH O-
OCH3
H
H
H
OCH3
H
2
3
-OCH O-
OCH3
OCH3
2
3i:
j:
H
H
-NHCOCH O-
OCH3
2
3
OCH3
OCH3 OCH3 -OCH CH O-
2 2
a
+
Reagents and conditions: (a) NaOH–EtOH, rt, 24 h; (b) PhI(OAc) , MeOH–H , rt, 8 h; (c)
2
+
+
EtOH–H , NH OHHCl, reflux, 2 h; (d) EtOH–H , N H 2HCl, reflux, 1 h; (e) 30% H O ,
2
2
4
2
2
1
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
NaOH–EtOH, rt, 48 h; (f) Et OBF , CH Cl , 0–20 C, 3h; (g) EtOH–H , reflux, 2 h; (h)
2
3
2
2
NiCl 6H O–NaBH , MeOH–THF, rt, 12 h.
2
2
4
Additional analogues of 1 and 3 that did not feature unprotected hydroxy moiety in the
benzene ring were prepared via a high-yield formylation of diarylethanones 22 followed by
condensation of the resulting intermediate 23 with NH NH or NH OH (Scheme 4). Of note,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
2
starting compounds 18 and 20 were efficiently prepared from the same benzaldehydes 11 (60–
0% yields). Although the described procedure included more stages, it allowed for both scale
9
up and improved total yields of the targeted compounds due to the isolation and purification of
intermediates 22 and 23.
Scheme 4. Synthesis of 4,5-Diarylpyrazoles 1 and 4,5-Diarylisoxazoles 3a
R1
O
O
O
R (R )
R2
R3
R2
R3
2
5
a
OH
b
Cl
R (R )
3
6
R (R )
R4
R4
4
7
O
1
1 R = H
17
18
R5
1
R2
f
c
+
R6
R3
R7
Cl
R5
R5
R5
R4
HO
Cl
Zn
d
e
22 60-90%
R6
R6
R6
R7
R7
R7
g
19
20
21
R2
R3
R4
R5
H
R6
R7
H
Yield
75%
O
R5
R2
1
1
o: OCH3 OCH3 OCH3
OCH3
p:
H
OCH3
H
OCH3
OCH3
OCH3 70%
R6
R3
N
R7
1q: OCH3 OCH3
Br
H
OCH3
H
H
H
79%
81%
R4
1
1
1
1
3
r: OCH3
s: OCH3
-OCH O-
H
H
OCH3
2
23 100%
-OCH CH O-
OCH3
46%
2
2
h
i
t:
H
H
OCH3
H
OCH3
-OCH O-
76%
2
u:
OCH3
H
OCH3
H
OCH3 66%
k: OCH3
OCH3
OCH3
H
OCH3
H
87%
HN N
O N
3l:
H
OCH3
H
OCH3 OCH3 OCH3 69%
R2
R3
R2
R3
3
3
3
3
3
m: OCH3 OCH3
Br
H
H
OCH3
OCH3
OCH3
H
H
H
64%
74%
n: OCH3
o: OCH3
-OCH O-
2
R4
R
4 R7
-OCH CH O-
H
72%
R7
R5
R5
2
2
R6
R6
p:
q:
H
H
OCH3
H
H
OCH3
-OCH O-
81%
1o-u
3k-q
2
OCH3
OCH3
H
OCH3 78%
a
65
Reagents and conditions: (a) CO(NH ) H O , CH OH, reflux, 1.5 h; (b) SOCl , CH Cl , drops
2
2
2
2
3
2
2
2
4
8
48
DMF, rt, overnight; (c) NaBH , MeOH, rt, 6–7 h; (d) SOCl , C H , 45–50 C, 40 min; (e)
4
2
6
6
6
6
66
Mg, ZnCl , LiCl, THF, rt, 2h; (f) CuCN2LiCl, THF, -20 C, 1 h, then rt overnight; (g)
2
1
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+
+
DMFDMA, without solvent, 60 C, 3h; (h) MeOH–H , N H 2HCl, reflux, 1 h; (i) MeOH–H ,
2
4
NH OHHCl, reflux, 2 h.
2
The same chemical route (Scheme 4) was reported in the literature to afford both
polymethoxy-4,5-diarylisoxazoles 3 and their 3,4-diarylsubstituted analogues 5 (Figure 2),67
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
8
however in our assessment the respective structures were not assigned properly. Considerably
less accessible 3,4-diaryl-5-unsubstituted isoxazoles 5a–c were selectively synthesized from
68
nitrostilbenes 25 and carbethoxypyridinium bromide. 3-Amino-4,5-diaryl- and isomeric 5-
amino-3,4-diarylisoxazoles 4 and 6 were accessed via the synthetic procedure published by our
team earlier.48, 51 Isoxazoles modified with the ortho-OH group (3r and 3s) were commercially
available from Chemical Block Ltd. Their structural integrity was confirmed prior to biological
testing.
To expand on a series of combretazoles, we prepared several reported combreta-1,2,3-
5
0
triazoles 7a–c. A more expeditious route to these molecules involving treatment of cyano-
5
2
combretastatins with NaN was published recently. The corresponding N-substituted isomers
3
50
8
a–c and 9a,b were synthesized as described earler. Compound structures are presented in
Table 5.
Finally, several combreta-3,4-diarylpyrroles 10a–e (Table 6) featuring substituents found
in the respective active analogues of pyrazole-, isoxazole- and 1,2,3-triazoles were
synthesized under conditions of the Barton-Zard reaction. Hydrolysis of the pyrrole-2-
carboxylates 25 followed by decarboxylation of the resulting pyrrole-2-carboxylic acids 26
afforded target 3,4-diarylpyrroles 10a–e (Scheme 5).
Scheme 5. Synthesis of 3,4-Diarylpyrroles 10a–ea
1
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
EtO C
HOOC
R1
2
O-
R1
R1
NH
NH
+
R2
N
R2
R2
+
C-
O
b
EtO CCH2
N
2
R3
R3
R3
a
R4
R4
R4
R5
2
4
25
26
R5
R5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
c
R1
H
R2
R3
R4
R5
Yield
H
N
R1
1
0a:
0b:
OCH3 OCH3 OCH3 OCH3 73%
OCH3 OCH3 OCH3 46%
-OCH O-
R2
1
1
1
1
H
H
0c: OCH3
0d:
0e: OCH3
OCH3
OCH3
60%
69%
60%
2
R3
H
OCH3 OCH3 OCH3
-OCH O-
H
R4
H
10a-e
2
OCH3
R5
a
Reagents and conditions: (a) K CO , EtOH, rt, 12–36 h; (b) (i) NaOH, EtOH–H O, reflux; 3–5
2
3
2
h; (ii) 18% HCl; (c) heating at mp, 1–2 min.
Biological Evaluation: SAR Studies
The phenotypic assessment of pyrazole, isoxazole, 1,2,3-triazole, and pyrrole derivatives
is summarized in Tables 3–6. Compounds CA4 and CA2 served as positive controls. In the sea
urchin embryo assay, diarylpyrazoles 1a, 1c, 1e, 1g–j, 1l, 1o–u, and 2a, diarylisoxazoles 3a–e,
3j–r, 4a–c, 5a, and 6, 4,5-diaryl-1,2,3-triazoles 7a and 7b, 1,5-diaryl-1,2,3-triazoles 8a and 8b,
and 3,4-diarylpyrroles 10a and 10b were determined to be potent antimitotic microtubule
destabilizing agents exhibiting profound cleavage alteration/arrest and embryo spinning.
Multiple derivatives including 1g–i, 1l, 1o, 1q–s, 1u, 3a–c, 3e, 3k, 3l, 3n, 3o, 6, and 7a showed
activity comparable to the parent CA4 and CA2. The most potent pyrazole 1p and triazole 7b
displayed higher antimitotic activity than that of combretastatins. Although compounds 1d, 1f,
1
m, 1n, 2b, 3i and 5b failed to induce embryo spinning, they were still classified as weak
microtubule destabilizing agents since the arrested post-treatment eggs aquired tuberculate
shape. Molecules 1b, 2c–e, 3g, 3h, 5c, 7c, 8c, and 9a showed antimitotic effects likely mediated
by non-tubulin mechanisms, whereas 1k, 9b, and 10c exhibited marginal antiproliferative
activity. Compounds 2f, 3f, 3s, 4d, 10d, and 10e failed to produce any activity up to 4 M
concentration.
1
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Table 3. Effect of Diarylpyrazoles 1 and 2 on Sea Urchin Embryos and Human Cancer
Cells
sea urchin embryo effects, EC (M)b
NCI60 screen
cleavage
alteration
0.002
cleavage
arrest
0.01
embryo
spinning
0.5
mean GI50,
compda
Mc
mean GI, %d
structure
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CA4
CA2
0.0032e
2.66f
0.002
0.01
0.5
HN
N
H CO
3
1
aa
H3CO
0.01
0.02
0.5
>5g
H CO
3
OH
OCH3
HN
N
1
b
H3CO
0.1
2
>10
2
NDh
OH
OCH3
N
H CO
3
NH
ca
0.007g
1
H3CO
0.05
0.2
H CO
3
OH
OCH3
N
NH
1d
H3CO
0.1
2 TEi
>10
NDh
OH
OCH3
H CO
N
3
NH
O
O
1
e
0.05
0.2
4
NDh
H CO
3
OH
OCH3
HN
N
H CO
3
1
fa
H3CO
0.05
0.5 TEi
0.05
>10
2
NDh
NDh
H CO
3
NO2
OCH3
N
NH
H CO
3
H CO
1
g
3
0.005
H CO
3
NO2
OCH3
1
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
HN
N
H CO
3
1
ha
H3CO
0.005
0.001
0.025
0.005
0.5
0.1
0.020
77.1
H CO
3
NH2
OCH3
N
NH
H CO
3
1
i
NDh
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H CO
3
H CO
3
NH2
OCH3
HN
N
H3CO
H3CO
NDh
1
j
0.5
4
2
1
73.0
H3CO
O
OCH3
HN
N
1k
1l
>4
>4
NDh
NDh
H3CO
O
OCH3
N
NH
H CO
3
0.005
0.1
0.02
1 TEi
1 TEi
0.05
0.5
>5
>5
0.5
H CO
3
H CO
3
O
O
N
H CO
3
NH
O
O
NDh
NDh
1m
H CO
3
OCH3
N
H
N
O
NH
1
n
O
0.2
H CO
3
OCH3
HN
N
H CO
3
1
o
H3CO
0.002
0.033
0.012
85.0
92.2
H CO
3
OCH3
N
H CO
NH
3
1
1
p
q
H CO
0.00025
0.005
0.005
0.02
0.05
0.1
3
H CO
3
OCH3
N
HN
H CO
3
NDh
H3CO
Br
OCH3
1
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HN
HN
N
H CO
3
1
r
O
0.005
0.005
0.01
0.05
0.05
0.2
0.5
0.5
2
0.309
0.182
87.2
77.0
O
OCH3
N
H CO
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
s
O
O
OCH3
N
NH
H CO
3
NDh
1
t
O
O
OCH3
N
NH
H CO
3
NDh
1
u
0.002
0.1
0.2
H CO
3
OCH3
H N
2
N
NH
H CO
3
2
aj
0.01
0.1
0.05
1 TEi
>4
1
0.046
89.2
75.8
54.0
H CO
3
H CO
3
OCH3
HN
N
H CO
3
NH2
2
bj
H3CO
>10
>10
0.302
H CO
3
OCH3
H N
2
N
NH
H CO
3
2cj
0.2
4.365
O
O
OCH3
HN
HN
N
H CO
3
NH2
dj
NDh
2
O
0.5
1
>4
>4
>5
>5
18.1
32.0
O
OCH3
N
H CO
3
NH2
2
ej
O
NDh
O
OCH3
1
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
H N
H CO
2
N
NH
3
O
2
fj
>4
>4
>4
NDh
O
H CO
3
OCH3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
53
69
48
Previously published compounds: 1a and 1c, 1f and 1h, 2a–f.
b
21
Hereinafter, the sea urchin embryo assay was conducted as described previously. 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.
c
Hereinafter, GI : concentration required for 50% cell growth inhibition.
5
0
d
e
f
Hereinafter, GI %: single dose inhibition of cell growth at 10 M concentration.
Data from ref. 70.
Mean value for 7 human cancer cell lines.71
g
h
i
53
GI for HUVEC.
5
0
Hereinafter, ND: not determined.
Hereinafter, TE: tuberculate eggs typical of microtubule destabilizing agents.
Synthesis, EC values and NCI60 screen data from ref. 48.
j
Table 4. Effect of Diarylisoxazoles 3–6 on Sea Urchin Embryos and Human Cancer Cells
sea urchin embryo effects, EC (M)
NCI60 screen
cleavage cleavage
embryo
compda
structure
alteration
arrest
spinning
mean GI, %
mean GI50, M
0.0009 HeLab
0.0014 HepG2b
0.0005 OVCAR-3b
O N
H CO
3
3
aa
H3CO
0.001
0.005
0.02
H CO
3
OH
OCH3
.0 HT29c
3
8.5 SVECc
N
O
0.0294 HeLab
H CO
3
3
ba
H3CO
0.002
0.002
0.005
0.02
0.05
0.05
0.0339 HepG2b
H CO
3
OH
.0148 OVCAR-3b
0
OCH3
N
O
H CO
3
3
c
ND
O
O
OH
OCH3
1
8
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N
O
H CO
O
3
O
3
d
0.05
0.002
>4
0.2
0.01
>4
4
ND
ND
ND
H CO
3
OH
OCH3
N
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3e
3f
H3CO
0.5
>4
OH
OCH3
N
O
H CO
3
O
O
H CO
3
OCH3
OCH3
N
H CO
3
O
O
O
3g
1
>4
>5
ND
H CO
3
OCH3
N
O
H CO
3
O
3
h
4
>4
>4
>5
ND
ND
O
H CO
3
H
N
N
O
O
3
i
O
0.2
4 TE
H CO
3
OCH3
N
O
H CO
3
H CO
3
j
3
0.02
0.1
2
ND
H CO
3
O
O
O
N
<0.01
86.7
92.3
H CO
3
0.0047 HeLab
3
ka
3
0.002
0.01
0.1
H CO
3
0.0044 HepG2b
H CO
OCH3
.010 OVCAR-3b
0
0
0.055
N
O
H CO
3
0
.299 HeLab
3
la
H3CO
0.005
0.1
0.5
H CO
0.396 HepG2b
3
OCH3
.223 OVCAR-3b
1
9
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O N
H CO
3
3
m
H3CO
0.01
0.005
0.002
0.02
0.05
0.1
0.2
0.2
0.2
2
ND
ND
Br
OCH3
O N
H CO
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
n
O
O
OCH3
O N
H CO
3
3
o
p
q
O
0.02
0.1
0.0549
2.455
88.0
O
OCH3
N
O
H CO
3
3
74.9
O
O
OCH3
N
O
H CO
3
3
0.15
0.2
>4
2
1
2
2
ND
H CO
3
OCH3
N
O
rd
OH
3.98
ND
65.0
4.3
3
H3CO
OCH3
N
O
H CO
3
sd
H CO
OH
>4
>4
3
3
OH
H N
2
N
O
H CO
3
4
ae
0.004
0.5
0.02
0.2
5
ND
H CO
3
H CO
3
OCH3
H N
2
N
O
H CO
3
4be
4
2.951
72.7
O
O
OCH3
2
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O
N
H CO
3
NH2
4ce
0.005
>4
0.02
>4
0.5
>4
ND
O
O
OCH3
H N
2
H CO
N
O
3
O
4
de
O
ND
2.7
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H CO
3
OCH3
O
N
H CO
3
5
af
H3CO
0.01
0.1
1
0.166
75.9
H CO
3
OCH3
O
N
H CO
3
5
bf
0.2
2
2 TE
>4
>10
>4
ND
ND
H CO
3
OCH3
O
N
H CO
O
3
5
cf
O
H CO
3
H N
2
O
N
H CO
3
6e
0.002
0.01
0.1
ND
H CO
3
H CO
3
OCH3
a
Previously published compounds: 3a,33, 34 3b, 3k and 3l, 4a–d and 6, 5a–c.
34
51
55
b
Data from ref. 34; HeLa: human cervical carcinoma, HepG2: human liver carcinoma, OVCAR-
: human ovarian carcinoma.
3
c
Data from ref. 33; HT29: human colon adenocarcinoma, SVEC: primary mouse seminal vesicle
epithelial cells.
d
Purchased from Chemical Block Ltd.
e
Synthesis, EC values and NCI60 screen data from ref. 51.
f
Synthesis, EC values and NCI60 screen data from ref. 55.
2
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 5. Effect of 1,2,3-Triazoles 7–9 on Sea Urchin Embryos and Human Cancer Cells50
sea urchin embryo effects, EC (M)
NCI60 screen
cleavage cleavage
alteration arrest
embryo
mean GI50,
compd
structure
spinning
M
mean GI, %
83.5
HN N
H CO
N
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
a
H3CO
0.005
0.0005
0.5
0.02
0.002
>4
0.1
0.05
>5
0.013
H CO
3
OCH3
HN
N
N
H CO
3
7
b
O
0.034
0.813
92.7
75.1
O
OCH3
H CO
HN N
3
O
N
O
7c
8a
H CO
3
OCH3
N
N
H CO
3
N
0
.027
H3CO
0.02
0.01
1
0.2
0.1
>4
>4
1
K562a
H CO
3
OCH3
N
N
H CO
3
N
8
b
O
0.5
>4
>5
0.178
83.8
O
OCH3
H CO
N
N
3
O
N
O
8
c
ND
H CO
3
OCH3
N
N
H CO
3
N
NH2
9
a
H3CO
0.1
ND
H CO
3
OCH3
N
H N
2
H CO
N
N
3
9
b
H CO
3
4
>4
>4
ND
9.9
H CO
3
OCH3
a
Data from ref. 37; K562: human myelogenous leukemia.
2
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Table 6. Effect of Diarylpyrroles 10 on Sea Urchin Embryos
sea urchin embryo effects, EC (M)
cleavage
alteration
cleavage
arrest
embryo
compd
structure
spinning
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NH
H CO
3
10a
H3CO
0.05
0.5
4
0.2
1
4
5
H CO
3
OCH3
NH
H CO
3
10b
H CO
3
OCH3
NH
H CO
3
O
O
1
0c
>4
>4
H CO
3
OCH3
NH
H CO
3
10d
10e
>4
>4
>4
>4
>4
>4
H CO
3
H CO
3
H CO
NH
3
O
O
H CO
3
In the series of pyrazoles featuring the 2,3,4-trimethoxyphenyl ring A next to the N atom
of pyrazole linker antitubulin activity decreased in the following order: 1o (4-methoxyphenyl
ring B) > 1h (3-amino-4-methoxyphenyl ring B) > 1a (3-hydroxy-4-methoxyphenyl ring B) >
1
f (3-nitro-4-methoxyphenyl ring B) > 1j (3-O-benzyl-4-methoxyphenyl ring B). Compounds
with trisubstituted rings A (1o, 1q, 1r, and 1s) displayed similar or equal effect, suggesting that
the presence of 3,4,5-trimethoxyphenyl ring A was not critical to the overall antimitotic effect. In
contrast, pyrazoles exhibiting 3-hydroxy-4-methoxyphenyl ring B next to the N atom (ex., 1c and
1
e) and tri- or tetrasubstituted ring A, exhibited more potent antitubulin effect as compared to
monosubstituted derivative 1d.
According to the literature, the replacement of 3-hydroxy group in the ring B of CA4 and
CA2 with 3-amino substituent afforded molecules with antiproliferative activity higher than the
parent CA2.71-73 In the sea urchin embryo assay, pyrazole analogues substituted with 3-amino
group in the ring B (1h, EC = 5 nM and 1i, EC=1 nM) exhibited activity similar to that of the
2
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1
1
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1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
parent CA4 (EC = 2 nM). Furthermore, these compounds were more potent than 3-
hydroxyphenyl derivatives 1a (EC = 10 nM) and 1c (EC = 50 nM), but less active than the
respective mono-methoxysubstituted pyrazoles 1o (EC = 2 nM) and 1p (EC = 0.25 nM). 3-
Nitrophenyl derivatives 1f (EC = 50 nM) and 1g (EC = 5 nM) were less potent.than their 3-
aminophenyl analogues 1h and 1i. Moreover, the removal of 3-hydroxy group from the ring B of
a and 1c furnished more potent compounds 1o, 1p. Notably, pyrazole 1l with disubstituted ring
B was more active than 1c.
The data further that the antimitotic activity of pyrazoles was not influenced by the 3-OH
0
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
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9
0
1
group in the ring B similar to the data reported in the literature.26, 27, 30 However, for less active
compounds containing tetrasubstituted ring A, namely 1e and 1m, lack of 3-hydroxy group
afforded less active molecule, 1e > 1m. Following these findings, pyrazole derivatives
substituted with 4-methoxyphenyl group (the B ring) were studied in more detail.
Compound 1p containing 3,4,5-trimethoxyphenyl ring A showed the best potency with
the EC value of 0.25 nM. The antimitotic antitubulin effect of compounds with different
substitution pattern in the A ring decreased in the following order: 1p > 1u (3,5-
dimethoxyphenyl ring A > 1t (3-methoxy-4,5-methylenedioxyphenyl ring A) > 1m (2,5-
dimethoxy-3,4-methylenedioxyphenyl ring A)  1b (3-hydroxy-4-methoxyphenyl ring A) > 1n
(1,4-benzoxazinone ring A) > 1k (3-O-benzyl- 4-methoxyphenyl ring A). Comparison of activity
for diarylpyrazole pairs featuring interchanged rings A and B (1a vs 1c, 1b vs 1d, 1f vs 1g, 1h vs
1i, 1o vs 1p, and 1r vs 1t, respectively) did not reveal any obvious trends: 1a > 1c (3,4,5-
trimethoxyphenyl and 3-hydroxy-4-methoxyphenyl rings); 1b = 1d (4-methoxyphenyl and 3-
hydroxy-4-methoxyphenyl rings); 1f << 1g (3,4,5-trimethoxyphenyl and 3-nitro-4-
methoxyphenyl rings); 1h < 1i (3,4,5-trimethoxyphenyl and 3-amino-4-methoxyphenyl rings);
1o << 1p (3,4,5-trimethoxyphenyl and 4-methoxyphenyl rings); 1r > 1t (3-methoxy-4,5-
methylenedioxyphenyl and 4-methoxyphenyl rings). Introduction of the 3-amino group in the
pyrazole ring significantly decreased antitubulin effect of the resulting compounds as shown
below (2a–f were considerably less potent than the respective unsubstituted molecules 1p, 1o, 1t,
1
r, 1s, and 1m).
In 4,5-diarylisoxazole series, removal of the 3-hydroxy group from the ring B decreased
antimitotic activity (3a vs 3k; 3b vs 3l; 3c vs 3p; 3d vs 3g). Moving a hydroxyl moiety from
position 3 (3e, EC = 2 nM) to 2-position was detrimental to the resulting effect, as shown by
compound 3r (EC = 200 nM). Molecule 3s with 2,4-dihydroxyphenyl substituent for the ring B
was inactive. Similarly, replacement of 3-hydroxy group with 3-methoxy functionality resulted
in a loss of activity (3d vs 3f). Agent 3b with 3-hydroxy-4-methoxyphenyl ring B was about 10
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times more active than 3j with 3,4-ethylenedioxyphenyl ring B, further suggestive of the
importance of 3-hydroxy group in the ring B for antimitotic antitubulin effect.
In 4,5-diarylisoxazoles with the ring A next to the O atom and 4-methoxyphenyl group
for the ring B the relative activity order was as follows: 3k (3,4,5-trimethoxyphenyl ring A) = 3o
(3-methoxy-4,5-ethylenedioxyphenyl ring A) > 3n (3-methoxy-4,5-methylenedioxyphenyl ring
A) > 3m (3,4-dimethoxy-5-Br ring A). Notably, compounds containing the 3,4,5-
trimethoxysubstituted ring A (3k, 3n, and 3o) showed consistently high activity with the EC
values of 2–5 nM for cleavage alteration. A replacement of 5-methoxy group of the ring A with
Br (3m) yielded a 10-fold reduction in potency. On the contrary, for isoxazoles featuring 3-
hydroxy-4-methoxyphenyl ring B next to the O atom as exemplified by 3b (3,4,5-
trimethoxyphenyl ring A), 3c (3-methoxy-4,5-methylenedioxyphenyl ring A) and 3e (4-
methoxyphenyl ring A) the EC values were similar (ca. 2–5 nM), however 3d (2,5-dimethoxy-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
,4-methylenedioxyphenyl ring A) was less active. We concluded that 2,3,4-tri- and 4-
monosubstituted ring A pharmacophores were well tolerated in these series, whereas tetra-
substituted aromatic ring yielded reduced antimitotic activity.
For 4,5-diarylisoxazoles with 4-methoxyphenyl pharmacophore for the ring B the
antimitotic activity decreased in the following order: 3l (3,4,5-trimethoxyphenyl ring A) > 3p (3-
methoxy-4,5-methylenedioxyphenyl ring A) > 3q (3,5-dimethoxyphenyl ring A) > 3i (1,4-
benzoxazinone ring A) > 3g (2,5-dimethoxy-3,4-methylenedioxyphenyl ring A). In our hands,
the derivative 3l with 3,4,5-trisubstituted ring A displayed the highest effect.
Changing positions of rings A and B in 4,5-diarylisoxazoles suggested that for the best
antimitotic activity, 3,4,5-trimethoxyphenyl or relevant group should be connected to position 5
of the isoxazole ring. Several representative examples included 3a > 3b (3,4,5-trimethoxyphenyl
and 3-hydroxy-4-methoxyphenyl rings), 3k > 3l (3,4,5-trimethoxyphenyl and 4-methoxyphenyl
rings), 3n > 3p (3-methoxy-4,5-methylenedioxyhenyl and 4-methoxyphenyl rings).
Shuffling N and O atoms in the isoxazole ring consistently led to a less active compound
(3l vs 5a; 3q vs 5b) indicating that 4,5-diarylisoxazoles 3 were more potent than their 3,4-
analogues 5. In both diarylisoxazole series 3 and 5, 3,4,5-trimethoxyphenyl substituent for the
ring A furnished the best activity. Molecules with 3,5-dimethoxyphenyl ring A were markedly
less potent than their respective 3,4,5-trimethoxyphenyl analogues, as shown by 3l vs 3q and 5a
vs 5b. Molecules 3h and 5c with unsubstituted ring B exhibited modest antiproliferative effect in
vivo.
Introducing unsubstituted NH group into the isoxazole ring (Figure 3) resulted in a
2
mixed outcome. For example, whereas amino-compounds of 4,5-diarylisoxazole series 4b (EC =
5
00 nM), 4c (EC = 5 nM), and 4d (EC > 4 M) were less potent than the respective analogues
2
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5
5
5
5
5
5
5
5
6
3
p (EC = 20 nM), 3o (EC = 2 nM), and 3g (EC = 1 M), compound 4a showed higher activity
than that of 3l. In contrast, 3,4-diarylisoxazole 6 (EC = 2 nM) exhibited enhanced activity
compared to 5a (EC = 10 nM). It is worth noting that for diarylisoxazoles introduction of the
amino group gave diverse results.
In summary, in the diarylisoxazole series the most active antimitotic compounds featured
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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7
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9
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3
4
5
6
7
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0
3
,4,5-trisubstituted phenyl pharmacophore for the ring A connected to position 5 of the isoxazole
ring. 3-Hydroxy-4-methoxyphenyl group for the ring B was essential. 4,5-Diarylisoxazoles were
more potent than their 3,4-analogues. Introduction of 3-amino group into 4,5-diarylisoxazoles
tended to reduce the activity whereas similar modification of the respective 3,4-diarylisoxazoles
improved antimitotic effect (6 vs 5a).
R
R
R
R
N
N
O
N
O
O N
H CO
O
O
3
H CO
O
H CO
3
R
3
H CO
H CO
N
3
3
H CO
O
O
3
H3CO
O
H CO
H CO
O
3
O
3
H CO
3
OCH3
OCH3
OCH3
OCH3
OCH3
3
l: R = H
3p: R = H
4b: R = NH2
3g: R = H
4d: R = NH2
3o: R = H
4i: R = NH2
5a: R = H
6: R = NH2
4a: R = NH2
Figure 3. Structures of aminoisoxazoles 4a-i, 6 and their unsubstituted analogues 3l, 3p, 3g, 3o,
and 5a.
In the diaryl-1,2,3-triazole series, the most potent molecules contained 3,4,5-
trimethoxyphenyl (7a and 8a) or 3-methoxy-4,5-methylenedioxyphenyl (7b and 8b )
pharmacophore as the ring A and 4-methoxyphenyl group as the ring B. Compounds with
tetramethoxy substituted ring A (7c and 8c) exhibited weak antiproliferative effect. The
introduction of 4-NH group into triazole linker of 1,5-diaryl-1,2,3-triazoles (8a vs 9a and 8b vs
2
9
b) markedly decreased antitubulin activity. 4,5-Diaryl-1,2,3-triazoles 7a and 7b (C–C
geometry) were found to be considerably more active than the respective 1,5-diaryl-1,2,3-
triazoles 8a and 8b (N–C geometry). We concluded that for the 1,2,3-triazole series, 3,4,5-
trimethoxyphenyl substituted the ring A enhanced the antitubulin effect, whereas 4-amino group
in the 1,2,3-triazole ring led to decreased activity. 4,5-Diaryl (C–C geometry) substitution
yielded more potent compounds.
3
,4-Diarylpyrroles were less potent than the parent CA2 and CA4. Compound 10a with
,4,5-trimethoxyphenyl ring A and 4-methoxyphenyl ring B was the most active derivative
displaying strong antimitotic microtubule destabilizing effect. Similar to isoxazoles, removal of
3
4-methoxy substituent from the ring A resulted in less active molecule 10b. Tetrasubstituted ring
A was not tolerated as compound 10c showed only marginal antiproliferative activity.
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Diarylpyrroles with unsubstituted ring B (10d and 10e) were inactive. In summary, for
diarylpyrrole series 3,4,5-trimethoxy substituted ring A and 4-methoxy group in the B ring were
essential for antitubulin activity.
Of combretazoles containing 3,4,5-trimethoxy substituted ring A and 3-hydroxy-4-
methoxy substituted ring B, isoxazoles 3 were more active than the respective pyrazoles 1
following the sequence 3a >> 1a; 3b > 1c; 3e >> 1d. Molecules exhibiting tetrasubstituted ring
A (ex., isoxazole 3d and pyrazole 1e) were equally potent. In a series of CA4 analogues
substituted with 3,4,5-trimethoxyphenyl pharmacophore as the ring A and 4-methoxyphenyl
group as the ring B the observed anti-mitotic activity varied as follows: 1p > 3k  1o > 7a  3l >
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
a > 8a > 10a (Figure 4).
Het
H CO
3
A
N
B
H CO
OCH3
3
OCH3
N
N
N
N
N
O
O
N
NH
NH
O
HN
NH
N
N
>

>

>
>
>
N
1
p
3k
1o
7a
3l
5a
8a
10a
Figure 4. Comparative activity of combretazoles with 3,4,5-trimethoxyphenyl ring A and 4-
methoxyphenyl ring B.
In general, 3,4,5-trimethoxyphenyl ring A was the optimal substituent, whereas
introducing tetra-substituted functionalities (2,5-dimethoxy-3,4-methylenedioxyphenyl)
significantly decreased the activity. In the respective series of combretazoles, 3,4,5-
trimethoxyphenyl-substituted molecules consistently displayed better antimitotic activity as
summarized below: pyrazoles: 1p >> 1m; 2a >> 2f; isoxazoles: 3l >> 3q; 4a >> 4d; triazoles: 7a
>
> 7c; 8a >> 8c; pyrroles: 10a >> 10c.
A summary of antimitotic activity for the combretazole derivatives substituted with
alternative pharmacophores as the ring A and 4-methoxyphenyl group as the ring B is shown in
Figure 5. As expected, both structure of the actual azole as well as nature of the ring A showed a
major impact on the relative activity of respective derivatives. In most cases, pyrazoles 1 were
more active than the corresponding isoxazoles 3. The same was observed for derivatives with
3,4,5-trimethoxyphenyl pharmacophore as the ring A and ethylenedioxyphenyl group as the ring
B: pyrazole 1l (EC = 5 nM) was more potent than the corresponding isoxazole 3j (EC = 20 nM).
4
,5(C–C)-Diaryltriazoles 7 displayed significant activity, whereas 1,5(N–C)-diaryltriazoles 8
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2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
were less active, and pyrroles 10 showed minimal effect. Of particular note was triazole 7b
endowed with 3-methoxy-4,5-methylenedioxy substituted ring A (EC = 0.5 nM). 4-Methoxy
group in the ring A of pyrazoles 1, isoxazoles 3 and 5, and pyrroles 10 was important to the
activity as follows: 1p > 1u; 3l > 3q; 5a > 5b; 10a > 10b.
Het
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
A
B
N
R
OCH3
Het
O
Ring A:
H CO
3
N
NH
NH
O
N
>
>
>
H CO
1u
3q
5b
10b
3
H CO
3
N
N
N
N
N
N
N
O
NH
HN
O
N
NH
O
>

>

>
N
O
O
7b
1r
3n
8b
1t
3p
H CO
3
N
N
HN
>
O
O
3
o
1s
H CO
3
N
N
N
N
NH
NH
N
NH
O
N
>
>

>
OCH3
N
O
O
1m
7c
3g
8c
10c
H CO
3
N
N
HN
O
>
H CO
3
Br
1q
3m
H
N
N
N
O
NH
O

O
1n
3i
H CO
3
Figure 5. Comparative activity of combretazoles with 4-methoxyphenyl group as the ring B.
In general, introduction of amino substituent into pyrazole and triazole fragment of the
combretazoles and to a lesser extent into isoxazole ring of 4,5-diarylisoxazoles decreased the
antimitotic antitubulin effect. However, 3,4-aminoisoxazole 6 with 3,4,5-trimethoxyphenyl ring
A and 4-methoxyphenyl ring B was as active as CA4 (EC = 2 nM), and 4,5-
diarylaminoisoxazole 4a showed comparable effect (EC = 4 nM). Similarly, introduction of the
amino group into the isoxazole ring of 4,5-diarylisoxazole 3o (EC = 2 nM) resulted in a modest
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change in activity (4c, EC = 5 nM). For aminoazole derivatives, isoxazoles 4 and 6 were more
active than respective pyrazoles 2 and 1,5-diaryltriazoles 9 (Figure 6).
NH2
Het
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
A
B
R
NH2
OCH3
Het
N
Ring A:
H CO
3
O
N
N
N
N
N
H N
H N
H N
NH2
NH2 H N
2
2
2
O
2
NH
HN
N
N
N
>
N
H CO
>
>
>

3
H CO
6
4a
2a
2b
9a
9b
3
H CO
3
N
N
H N
H N
2
2
NH
O
>
O
O
2c
4b
H CO
3
N
N
NH2
NH2
O
>> HN
O
O
4c
2e
Figure 6. Comparative activity of combretazoles with amino group in N-heterocycle.
The sea urchin embryo data for the antitubulin compounds correlated well with the
respective assays using human cancer cell lines (Tables 3–5). For example, the most potent
pyrazole derivative identified in vivo (1p) showed the highest cytotoxicity in NCI60 screening.
Similarly, compounds 1h, 1o, 1r, and 1s displaying good activity in the sea urchin embryos also
exhibited sub-M GI values in the cell-based screening. Pyrazole 1h with 3-amino group in the
5
0
ring B altered cleavage at 5 nM concentration in the sea urchin assay while exhibiting the IC50
6
9
value of 8.4 nM in murine adenocarcinoma colon 26 cells. In a similar fashion, compounds 2c,
d, and 2e identified as weaker hits in vivo were also less cytotoxic against human cancer cells.
Aminopyrazoles were generally less cytotoxic than their unsubstituted analogues (2a < 1p; 2b <
o; 2d < 1r; 2e < 1s).
In the sea urchin embryo model, pyrazole 1a with trimethoxyphenyl ring A next to the
2
1
N1 atom showed enhanced activity compared to its analogue 1c (EC values of 10 and 50 nM,
respectively) (Table 3). In contrast, 1a failed to inhibit HUVEC (human umbilical vein
endothelial cells) growth up to 5 M concentration, although the reported HUVEC cytotoxicity
2
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