Pyrrole-based antitubulin agents: Two distinct binding modalities are predicted for C-2 analogues in the colchicine site

Article abstract of DOI:10.1021/ml200217u

3,5-Dibromo-4-(3,4-dimethoxyphenyl)-1H-pyrrole-2-carboxylic acid ethyl ester is a promising antitubulin lead agent that targets the colchicine site of tubulin. C-2 analogues were synthesized and tested for microtubule depolymerizing and antiproliferative activity. Molecular modeling studies using both GOLD docking and HINT (Hydropathic INTeraction) scoring revealed two distinct binding modes that explain the structure-activity relationships and are in accord with the structural basis of colchicine binding to tubulin. The binding mode of higher activity compounds is buried deeper in the site and overlaps well with rings A and C of colchicine, while the lower activity binding mode shows fewer critical contacts with tubulin. The model distinguishes highly active compounds from those with weaker activities and provides novel insights into the colchicine site and compound design.

Full text of DOI:10.1021/ml200217u

Letter
pubs.acs.org/acsmedchemlett
Pyrrole-Based Antitubulin Agents: Two Distinct Binding Modalities
Are Predicted for C-2 Analogues in the Colchicine Site
Chenxiao Da,† Nakul Telang,‡ Peter Barelli,‡ Xin Jia,‡ John T. Gupton,‡ Susan L. Mooberry,§
and Glen E. Kellogg*,†
†Department of Medicinal Chemistry & Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University,
Richmond, Virginia 23298-0540, United States
‡Department of Chemistry, Gottwald Center for the Sciences, University of Richmond, Richmond, Virginia 23173, United States
§Department of Pharmacology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900, United
States
S
* Supporting Information
ABSTRACT: 3,5-Dibromo-4-(3,4-dimethoxyphenyl)-1H-pyrrole-2-carboxylic acid ethyl ester is a
promising antitubulin lead agent that targets the colchicine site of tubulin. C-2 analogues were
synthesized and tested for microtubule depolymerizing and antiproliferative activity. Molecular modeling
studies using both GOLD docking and HINT (Hydropathic INTeraction) scoring revealed two distinct
binding modes that explain the structure−activity relationships and are in accord with the structural basis
of colchicine binding to tubulin. The binding mode of higher activity compounds is buried deeper in the
site and overlaps well with rings A and C of colchicine, while the lower activity binding mode shows fewer critical contacts with
tubulin. The model distinguishes highly active compounds from those with weaker activities and provides novel insights into the
colchicine site and compound design.
KEYWORDS: Antitubulin, hydropathic interactions, docking, multifunctional pyrroles, structure−activity relationship
the colchicine site.8 This structural diversity provides many
icrotubules are major cytoskeletal components in
possibilities for optimization and new scaffold design. The
Meukaryotic cells and participate in a variety of cell
colchicine site has been characterized with X-ray crystallog-
raphy by cocrystallization of the protein with DAMA-
colchicine;9 the site is at the interface between α- and β-
tubulin. Complexation with other agents has documented the
flexibility of this pocket.10 To understand the structural basis
for ligand binding at the colchicine site, a common
pharmacophore model was built by Nguyen et al. based on
15 structurally diverse colchicine site inhibitors.11
functions including maintenance of cell shape, intracellular
transport, and forming mitotic spindles for segregating
chromosomes during mitosis. Microtubules assemble and
disassemble by a reversible process called dynamic instability
involving discrete α/β tubulin heterodimers.1 Diverse agents
suppress microtubule dynamics; in rapidly dividing cells, they
induce mitotic arrest and initiate apoptosis.2 Compounds that
target microtubules bind at four major binding sites: the taxane
and the laulimalide/peloruside A sites for microtubule-
stabilizing agents and the vinca and colchicine sites for
microtubule-destabilizing agents.2,3 Taxanes and vinca alkaloids
have achieved notable success in cancer chemotherapy, but no
colchicine site agents have been approved for systemic use
against cancer.4
We previously showed that 1 [3,5-dibromo-4-(3,4-dimethox-
yphenyl)-1H-pyrrole-2-carboxylic acid ethyl ester, JG-03-14,
Figure 1] is a potent microtubule-destabilizing agent.12 Because
Recent studies of one family of colchicine site agents,
analogues of combretastatin A4 (CA4), have reported
antivascular actions leading to the rapid collapse of tumor
vasculature.5 A number of CA4 analogues are in clinical trials
refueling the search for novel colchicine site agents. Emerging
drug resistance due to the expression of the βIII-tubulin isotype
has compromised the clinical use of taxanes and vinca
alkaloids.6 Resistance to different types of microtubule targeting
agents was recently suggested to be related to their binding
sites, and that βIII-tubulin mediated drug resistance might be
circumvented by colchicine site agents.7 Natural and synthetic
compounds, for example, podophyllotoxins, arylindoles,
sulfonamides, 2-methoxyestradiols, and flavonoids, bind within
Figure 1. Structures of colchicine and lead compound JG-03-14 (1).
Received: September 9, 2011
Accepted: November 1, 2011
Published: November 1, 2011
© 2011 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
Scheme 1. Preparation of JG-03-14 Analogues with Modifications at the C-2 Position; See Table 1 for Identities of Various R
a
Groups
a
Reagents: (a) POCl3, DMF and heat, followed by H2O/NaPF6. (b) Glycine ethyl ester or glycine t-butyl ester and NaOt-Bu, DMF, and heat. (c)
NaOH, EtOH/H2O, and heat. (d) ROH, 1,1′-carbonyldiimidazole, DBU, and DMF. (e) Dibromodimethylhydantoin, CHCl3, and heat.
Table 1. Structures, Biological Activity, and Properties of Pyrrole Compounds 1 and 7a−l
a
b
c
d
compd
R
antiproliferation IC50 (μM) cellular microtubule loss
binding mode HINT score
549
HINT log P ALOGPs
colchicine
0.016 ± 0.002
0.036 ± 0.002
0.618 ± 0.07
0.067 ± 0.002
0.109 ± 0.008
1.82 ± 0.3
1.30 ± 0.04
3.3 ± 0.3
5.3 ± 0.3
4.6 ± 0.2
5.2 ± 0.3
8.0 ± 0.3
100% loss at 0.5 μM
100% loss at 0.5 μM
50% loss at 5 μM
3.24
2.60
2.06
3.14
3.14
3.24
3.68
4.76
3.61
2.52
2.57
0.27
0.78
4.37
1.59
4.44
3.87
4.74
4.70
5.02
5.05
5.83
5.39
4.10
3.82
0.39
0.27
5.48
e
1
ethyl
I
418
524
157
−179
187
530
256
713
293
358
631
774
957
7a
7b
7c
7d
7e
7f
methyl
n-propyl
i-propyl
t-butyl
n-butyl
n-hexyl
benzyl
I
75% loss at 5 μM
I
70% loss at 5 μM
I
no loss up to 10 μM
15% loss at 10 μM
35% loss at 10 μM
no loss up to 10 μM
10% loss at 10 μM
10% loss at 10 μM
no loss up to 10 μM
no loss up to 10 μM
II
II
II
II
II
II
II
II
II
7g
7h
7i
−(CH2)3NMe2
−(CH2)2NMe2
7j
−(CH2)3NMe2H+Cl−
−(CH2)2NMe2H+Cl−
4-methoxylphenyl
7k
7l
10.7 ± 0.4
18.3 ± 2.7
no loss up to 10 μM
a
b
c
Experiments were performed using human MDA-MB-435 cancer cells. Loss of interphase microtubules was evaluated in A-10 cells. 515 HINT
d
e
score units ≈ 1 kcal mol−1 (ref 15). ALOGPs were calculated at Virtual Computational Chemistry Laboratory, http://www.vcclab.org. Ref 12.
1 inhibited the binding of [3H]colchicine and COMPARE
analysis, which evaluates the similarity between two compounds
with respect to the NCI 60-cell line assay,13 showed correlation
between 1 and colchicine, it is likely that 1 also binds at this
site.14 Although 1 does not structurally resemble other classes
of agents, it more or less fits the previous pharmacophore
model.14 In addition, 1 and an unfocused set of analogues
produced a quantitative linear quantitative structure−activity
relationship (QSAR) relationship between the IC50 and the
HINT15 binding score.14 This scoring model, which considers
hydrophobic and polar interactions as well as entropic effects,
has been shown to correlate with binding free energy for small
molecule−biomacromolecular complexes.16
suggest that it is a viable lead candidate for optimization as a
new colchicine site anticancer agent.17−19 Of particular note is
that there is considerable synthetic flexibility for 1 and
analogues, such that each of the five atoms of the pyrrole
ring can be differentially probed, elaborated, and optimized for
SAR. Here, we report the synthesis, physical properties, and
microtubule inhibitory effects for C-2 analogues of 1. Modeling
studies indicate that two distinct binding modalities are
required to explain the observed SAR.
In this study, we retain the 3,4-dimethoxylphenyl at C-4 and
the two bromine groups at C-3 and C-5 of 1 and focus on
modifications to the ester at the C-2 position of the pyrrole
core. We have previously reported17 the synthesis of 1 (JG-03-
14) and have utilized a similar sequence of reactions as outlined
in Scheme 1 to prepare the new analogues listed in Table 1. 3,4-
Further investigations on autophagic cell death, polyploidy,
senescence, and effects on endothelial cell functions for 1
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ACS Medicinal Chemistry Letters
Letter
Dimethoxyphenylacetic acid (2) was converted to the
corresponding vinamidinium salt (3) using Vilsmeier−
Haack−Arnold conditions. Compound 3 was condensed with
glycinate esters to give either pyrrole ethyl ester (4a) or pyrrole
t-butyl ester (4b). Compound 4a was hydrolyzed with base in
aqueous ethanol to produce the corresponding pyrrole acid (5),
which served as the key building block for the majority of the
analogs. The various pyrrole esters (6a−i) were constructed
with the appropriate alcohol, carbonyldiimidazole, DMF, and
DBU. The final step involving dibromination was accomplished
with dibromodimethylhydantoin in refluxing chloroform. The
only exception was converting the pyrrole t-butyl ester (4b)
directly to the corresponding dibromopyrrole (7) with
dibromodimethylhydantoin. All reactions gave product yields
in excess of 65%.
Figure 2. Colchicine (yellow) and binding modes of pyrrole-based C-2
analogues (mode I, red; mode II, purple). The extents of the
colchicine site, as illustrated by MOLCAD, are shown in grayish white.
Antiproliferative activities were measured in MDA-MB-435
cancer cells using the sulforhodamine B assay, and effects on
cellular microtubules were evaluated in A-10 cells using
immunofluorescence as previously described.12 Results are
presented in Table 1.
the subpocket A, overlapping the trimethoxyphenyl ring (ring
A) of DAMA-colchicine. The positions of the C-2 ester chain
differ between the two modes. In mode I, the R group of the
ester has “acceptable” size (i.e., 1 and 7a−c) and fits within
subpocket C and thus overlaps well with ring C of DAMA-
colchicine, while in mode II, the bulkier 7d−l R groups extend
out from the main pocket toward its opening.
To illustrate the specific interactions between the ligands and
the site, we calculated intermolecular HINT interaction maps21
using 1 as representing mode I binding (Figure 3A) and 7e
For this study, the SAR was analyzed with respect to the
antiproliferative activities of compounds 1 and 7a−l. The
antitubulin activity generally trends with antiproliferative
activity. Compound 1 remains the most active compound (36
nM).12 As compared to 1, 7a had a 17-fold decrease in activity
likely due to its one-carbon shorter ester. Similarly, the longer
and bulkier alkyl substitutions n-propyl (7b) and i-propyl (7c)
decreased antiproliferative activity. Larger groups, t-butyl (7d),
n-butyl (7e), or n-hexyl (7f), were tolerated but with a
significant activity loss of at least 36-fold. A dramatic loss was
also observed for aromatic substitutions (7g and 7l). The
incorporation of a comparatively polar amine did not increase
the activity significantly (7h−k), suggesting that the activity
drop is truly related to sterics and not solubility.
The observation that the protonated amines (7j and 7k) had
a further 2-fold drop in activity as compared to their free base
analogues (7h and 7i) may be due to their weaker ability to
penetrate the cell membrane. Moreover, no microtubule effects
were observed up to 10 μM for the amine derivatives,
suggesting that a different mechanism of action of antiprolifera-
tion might be at play. The SAR suggests that only the properly
sized group would be favorable for activity, and the ethyl group
of 1 provides that optimum.
To rationalize the SAR from a structure-based perspective,
we performed docking studies with the X-ray crystal structure
of DAMA-colchicine/tubulin (PDB ID: 1sa0).9 It should be
noted that the resolution of the 1sa0 structure for αβ-tubulin is
poor (3.58 Å), and resulting modeling studies have a higher
degree of uncertainty than in other systems. The colchicine site
is mostly buried in the β-subunit surrounded by helices H7 and
H8, loop T7, and strands S8 and S9. The T5 loop of the α-
subunit also contributes to the pocket (see Figure 2). DAMA-
colchicine occupies the pocket such that ring A fits deep within
a subpocket close to H7, ring C fits into another subpocket
close to T5, ring B is centered within the main pocket, and the
DAMA chain is pointing to the pocket's entrance. For
convenience, we will refer to the subpockets where rings A
and C bind as subpockets A and C. Compound 1 and its
analogues 7a−l were docked in the colchicine site with poses
generated by the docking program GOLD20 and rescored with
HINT.15,16 The compounds can be divided into two sets based
on their computationally predicted binding modes (Table 1 and
Figure 2). In both modes, the dimethoxyphenyl ring locates in
Figure 3. HINT interaction maps of (A) 1 (binding mode I) and (B)
7e (binding mode II). For 1 and 7e, green contours represent
favorable hydrophobic interactions; blue contours represent favorable
polar interactions (hydrogen bonds, acid/base, Coulombic); red
contours represent unfavorable polar interactions. Compound 1 is
shown in red, 7e is shown in purple, and colchicine is shown in yellow.
representing mode II (Figure 3B). First, subpocket A, which fits
the dimethoxyphenyl ring in both modes, is quite hydrophobic.
In both modes, the four-carbon side chains of Leu248β and
Leu255β clamp the phenyl ring in place, while deeper in the
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ACS Medicinal Chemistry Letters
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pocket other residues lock the ligand's methoxys. Polar
interactions also play a part as Cys241β is in proximity to
these two methoxys, with distances between the cysteine's
sulfur and the oxygens of 3.06 and 3.45 Å, thus likely forming at
least one hydrogen bond to support the binding. Also in both
modes, there is a favorable interaction in the main pocket
between the backbone oxygen of Asn258β and the ligand's
pyrrole nitrogen.
Both hydrophobic and polar residues characterize subpocket
C, which fits the esters in mode I binding. The alkyl ends reach
the hydrophobic bottom, while the carboxyl oxygens anchor the
ester by forming hydrogen bonds with the backbone nitrogen
of Val181α. The main pocket includes its funnel opening and is
much more spacious than subpocket C. It easily tolerates the
size of the longer esters binding with mode II by flipping the
pyrrole core, thus exposing the ester tail to the solvent while
keeping the dimethoxylphenyl ring in subpocket A. Our models
suggest that a new hydrogen bond, stabilizing the ester tail in
mode II, is formed between the amide nitrogen of Asn101α and
the ligand's carbonyl oxygen. The interactions for the various R
groups of 7d−l are poorly defined as the pocket entrance
broadens and has a large solvent exposure.
Table 2. HINT Scores by Fragment and Interaction Type
a
HINT score
ring
ester
compd mode HTOTAL HHB + HAB
HHH HAA + HBB
HHP
1
I
492
518
363
321
570
626
435
589
557
581
650
596
587
858
680
1069
766
778
781
665
812
549
726
375
847
827
711
583
855
833
226
225
387
284
268
237
213
196
494
−499
−427
−593
−478
−398
−364
−369
−341
−336
−400
−236
−403
−240
−1328
−1048
−1681
−1821
−1169
−1015
−1046
−868
−1071
−1027
−634
−755
−924
7a
7b
7c
7d
7e
7f
I
I
I
II
II
II
II
II
II
II
II
II
7g
7h
7i
7j
7k
7l
a
Interaction types: favorable polar (hydrogen bond, HHB, and acid/
base, HAB), hydrophobic (HHH), unfavorable polar (acid/acid, HAA
and base/base, HAB), and unfavorable hydrophobic polar (HHP).
,
The compounds in mode I displayed notably higher
antiproliferative activity and antitubulin activity than the
compounds in mode II. It is clearly important to effectively
occupy both subpockets A and C in the colchicine site. The
SAR within the mode I set is size related: The methyl of 7a, the
n-propyl of 7b, and the i-propyl of 7c may not position the
ester carbonyl (hydrogen bonded to Val181β) as well as the
ethyl of 1. In contrast, in the mode II set, the ester R extends
from the pocket into (and possibly out of) the pocket's
entrance. The SAR simply may not be interpretable as these
tails are highly flexible and thus subject to interactions with a
wide array of residues as well as solvent.
It is also instructive to compare, in detail, the binding of
colchicine and the pyrrole-based compounds 1 and 7a−l: (1)
depletion of ring B of colchicine retains activity, while rings A
and C, which adopt a similar conformation as in mode I, are
necessary for high affinity binding;22 (2) residues Cys241β
(subpocket A) and Val181α (subpocket C) appear to be
important for antitubulin activity since the removal of any A
ring methoxy group close to Cys241β weakens the binding to
tubulin and microtubule inhibition.23 Also, isocolchicine, whose
structural difference to colchicine is in the C ring (methoxy at
C-9 and keto at C-10) binds weakly and only poorly inhibits
microtubule assembly,24 probably because of a loss of hydrogen
bonding to Val181α. Both residues anchor the ligand in the
more active mode I, while only Cys241β does so in the less
active mode II. This may largely explain the difference in
activity between the binding modes.
The HINT scores of Table 1 were poor in distinguishing
between binding in modes I and II. The reasons for this failure
are instructive. First, the poor resolution of the tubulin crystal
structure and the flexibility of the pocket,10 especially the T5
and T7 loops, are a partial explanation. However, the binding
modes themselves and the nature of the pocket are larger
factors. Table 2 lists the HINT scores in terms of two
fragmentsthe common dimethoxyphenyl plus pyrrole (ring)
and the ester. Interaction types further differentiate the latter.
The total ring score is largely invariant (580 ± 70), excluding
7b and 7c, where it is lower by >200. The ester's HHH for mode
I (750 ± 130) is much higher than for mode II (280 ± 90).
Interestingly, HHH is highest for 7b and 7c, but accommodation
of these longer esters was penalized by poorer ring interactions.
For 1 and 7a−c, hydrophobic binding quality in subpocket C is
key. Although the esters of mode II compounds appear to make
productive contacts, these are in the very open funnel-like
entrance of the pocket where dynamic solvent effects that can
disrupt polar interactions must be assumed.
In summary, mode I is a new binding motif observed for
pyrrole compounds based on JG-03-14 (1) that is different
from previously reported binding modes.11,14 The ester chain in
mode I overlaps with the C-10 substituents of colchicine and
the SAR of colchicine C-10 analogues also shows that
increasing length of the alkyl chain causes a concomitant
decrease in activity.25 We propose that the deeper burial of
mode I ligands is more disruptive to the association of α- and β-
tubulin subunits than is binding with mode II. We are
continuing design and development of additional JG-03-14
(1) analogues by focusing on other positions of the pyrrole
core as we attempt to gain a full view of the SAR.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details for the synthesis and characterization of
reported compounds, procedures for the assays, and additional
information on the computational methods utilized. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 804-828-6452. E-mail: glen.kellogg@vcu.edu.
Funding
This work was partially supported by NIH R01 CA135043 (to
D. A. Gewirtz, VCU), NIH R15 CA067236 (to J.T.G.), and the
President's Council Research Excellence Award (to S.L.M).
ACKNOWLEDGMENTS
■
The excellent technical assistance of Lyda Robb, Cara
Westbrook, and Nicholas Dbydal-Hargreaves is gratefully
acknowledged.
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Cabot, M. C.; Newsham, I. F.; Gewirtz, D. A. Autophagic Cell Death,
Polyploidy and Senescence Induced in Breast Tumor Cells by the
Substituted Pyrrole JG-03−14, a Novel Microtubule Posion. Biochem.
Pharmacol. 2007, 74, 981−991.
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