Kinesin spindle protein inhibitors with diaryl amine scaffolds: Crystal packing analysis for improved aqueous solubility

Article abstract of DOI:10.1021/ml500016j

Diaryl amine derivatives have been designed and synthesized as novel kinesin spindle protein (KSP) inhibitors based on planar carbazole-type KSP inhibitors with poor aqueous solubility. The new generation of inhibitors was found to show comparable inhibitory activity and high selectivity for KSP, and this was accompanied with improved solubility. Kinetic analysis and molecular modeling studies suggested that these inhibitors work in an ATP-competitive manner via binding to the secondary allosteric site formed by α4 and α6 helices of KSP. Comparative structural investigations on a series of compounds revealed that the higher solubility of diaryl amine-type inhibitors was attributed to fewer van der Waals interactions in the crystal packing and the hydrogen-bond acceptor nitrogen of the aniline moiety for favorable solvation.

Full text of DOI:10.1021/ml500016j

Letter
pubs.acs.org/acsmedchemlett
Kinesin Spindle Protein Inhibitors with Diaryl Amine Scaffolds:
Crystal Packing Analysis for Improved Aqueous Solubility
Tomoki Takeuchi,† Shinya Oishi,*,† Masato Kaneda,† Hiroaki Ohno,† Shinya Nakamura,‡
Isao Nakanishi,‡ Masayoshi Yamane,§ Jun-ichi Sawada,§ Akira Asai,§ and Nobutaka Fujii*,†
†Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
‡Faculty of Pharmacy, Kinki University, 3-4-1 Kowakae, Higashi-osaka 577-8502, Japan
§Graduate School of Pharmaceutical Sciences, University of Shizuoka, Suruga-ku, Shizuoka 422-8526, Japan
S
* Supporting Information
ABSTRACT: Diaryl amine derivatives have been designed and synthesized as novel kinesin
spindle protein (KSP) inhibitors based on planar carbazole-type KSP inhibitors with poor aqueous
solubility. The new generation of inhibitors was found to show comparable inhibitory activity and
high selectivity for KSP, and this was accompanied with improved solubility. Kinetic analysis and
molecular modeling studies suggested that these inhibitors work in an ATP-competitive manner via
binding to the secondary allosteric site formed by α4 and α6 helices of KSP. Comparative
structural investigations on a series of compounds revealed that the higher solubility of diaryl
amine-type inhibitors was attributed to fewer van der Waals interactions in the crystal packing and
the hydrogen-bond acceptor nitrogen of the aniline moiety for favorable solvation.
KEYWORDS: Aqueous solubility, crystal packing, diphenylamine, kinesin spindle protein
he kinesin spindle protein (KSP; also known as Eg5) is a
employed for in vivo studies. To overcome the inherent
Tmember of the kinesin superfamily of molecular motor drawbacks of carbazole-based KSP inhibitors, we undertook
proteins.1 The KSP moves toward the plus end of microtubules
research on the development of novel diaryl amine-type KSP
inhibitors to simultaneously satisfy the potent inhibitory activity
as well as show much better solubility in aqueous solution. The
structural basis of the solubility of a series of compounds was
also investigated by single-crystal X-ray diffraction studies and
free energy calculations.
using the energy generated from ATP hydrolysis. This KSP
movement produces the outward force that pushes the
centrosomes apart during cell division and provides the
following formation of the bipolar spindle. Inhibition of KSP
prevents spindle pole separation, which leads to prolonged
mitotic arrest in prometaphase and subsequent apoptosis.2
Unlike tubulin and microtubules, KSP expression is abundant
only in dividing cells, but not in postmitotic neurons in the
human central nervous system.3 Therefore, KSP inhibitors are
expected to be more favorable agents for cancer chemotherapy
without the neurotoxic side effects seen with traditional
antimitotic agents (e.g., taxanes and vinca alkaloids).4−6 To
date, several clinical trials of potent KSP inhibitors including
ispinesib, SB-743921, AZD4877, ARRY-520, and 4SC-205 have
been conducted.1
Recently, we reported that carbazole derivative 3 exhibited
potent KSP inhibitory activity.7 On the basis of the common
substructure of the known KSP inhibitory terpendole E 1 and
HR22C16 2 (Figure 1),8,9 the ring-fused indoles were identified
to be minimal scaffolds for KSP inhibition. Further structure−
activity relationship studies from carbazole 3 in combination
with the known biphenyl-type KSP inhibitors like 410−12
revealed that a carboline 5 and a lactam-fused carbazole 6a
exhibited potent KSP ATPase inhibitory activity and cytotox-
icity via effective cell-cycle arrest at the M-phase.13 During the
course of our investigations on antitumor effects of these
carbazole-based KSP inhibitors,14,15 we found that these
inhibitors exhibited limited solubility in aqueous solvents
The melting points of carbazole-type KSP inhibitors 6a,b
were extremely high (Figure 1). We speculated that the poor
solubility of compound 6 would be attributable to the
significant intermolecular interactions in the crystals since the
melting point is correlated with the crystal packing of the
molecule, which is one of the major contributing factors to
solubility.16 With the aim of disrupting the possible
intermolecular π−π stacking interactions to lower the melting
point and in turn to improve the solubility, the design of more
nonplanar analogues from planar compounds 6 was expected to
be a promising approach.16 Alternatively, the addition of polar
or ionizable functional group(s) is also an effective modification
to enhance solubility. To satisfy these two criteria, we designed
diphenylamine derivatives 7a,b, in which the pyrrole C−C
bond in the central part of carbazoles 6a,b was cleaved (Figure
1). It was expected that the two aryl rings in potentially
noncoplanar conformations in 7 would prevent intermolecular
π−π stacking interactions and that the newly available aniline
would enhance solubility in an aqueous environment.
Received: January 16, 2014
Accepted: March 10, 2014
Published: March 10, 2014
© 2014 American Chemical Society
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position exhibited four times more potency (IC50 = 0.045 μM)
than the parent carbazole 6b. This potency was comparable to
that of the most potent carbazole-type inhibitor 6a. Diphenyl-
amine 7b showed a good inhibitory effect on the proliferation
of cancer cell lines: A549, HCT-116, and MCF-7 (see
Supporting Information).
To investigate the selectivity of diphenylamine 7b, inhibitory
activities were evaluated for a number of the other kinesins.
The ATPase activities of centromere-associated protein E
(CENP-E), Kid, mitotic kinesin-like protein 1 (MKLP-1),
KIF4A, KIFC3, KIF14, and KIF2A were not inhibited by 7b at
20 μM (see Supporting Information), which is consistent with
the selectivity profile of carbazole-type inhibitor 6a. The
diphenylamine 7b was also screened in comparison with β-
carboline 5 and carbazole 6a against a panel of 117 kinases to
assess the selectivity profiles (see Supporting Information). β-
Carboline 5 showed >50% inhibition against four kinases
(CDK6/cyclinD3, IKKβ, PRK2, and ROCK-II) at 10 μM,
which is consistent with previous studies showing that carboline
is a well-known scaffold of kinase inhibitors.18 However, no
more than 50% inhibition against any kinases was observed in
6a and 7b (10 μM), suggesting that newly identified
diphenylamine-type inhibitor 7b has high selectivity for KSP.
To estimate the possible binding pocket of the diaryl amine-
type inhibitor 7b, kinetic experiments of KSP ATPase were
conducted as a function of ATP concentration in the absence
or presence of 7b (see Supporting Information). Lineweaver−
Burk plots revealed that the maximum velocity (Vmax) value in
the presence of 7b was identical to that in the absence of 7b.
This suggested that diphenylamine 7b inhibited KSP ATPase
activity in an ATP-competitive manner as observed for biphenyl
4, β-carboline 5, and carbazole 6a,13 possibly by binding to the
ATP binding site or the α4/α6 allosteric site.11
To further provide insights into the binding site of diaryl
amine-type KSP inhibitors, possible interactions of diphenyl-
amine 7b to three possible binding sites (i.e., the ATP binding
site, the α2/α3/L5 allosteric site, and the α4/α6 allosteric site)
were estimated by a simulated docking study using the MM/
GBVI method. The predicted binding affinity (Ebind) of 7b to
the α4/α6 allosteric site (−24.7 kcal/mol) was higher than the
values computed to the other sites (−20.3 kcal/mol for the
ATP binding site; −19.7 kcal/mol for the α2/α3/L5 allosteric
site). In addition, the high selectivity of 7b for KSP compared
with any other kinesins and kinases also supports the unlikely
affinity to the ATP binding pocket, which is highly conserved
among kinesins.19
Figure 1. Structures of the reported KSP inhibitors 1−6 and design of
novel KSP inhibitors 7 with a diphenylamine scaffold.
A series of diaryl amine derivatives 7 and 8 were prepared by
palladium-catalyzed N-arylation using aryl bromides and
substituted anilines (see Supporting Information).17 The
diphenylamine derivatives 7a,b were initially evaluated for
KSP ATPase inhibitory activity (Table 1). Diphenylamine 7a
with the accessory amide group at the 3-position on the left-
hand phenyl group showed no KSP inhibitory activity; although
the parent carbazole 6a showed highly potent activity.
However, diphenylamine 7b with the amide group at the 4-
Table 1. KSP Inhibitory Activities of Diphenylamines with a
3,4-Fused Lactam Structure on the Left-Hand Phenyl Group
and the Related Carbazoles
The docking pose of diphenylamine 7b in the pocket formed
by helices α4 and α6 is shown in Figure 2. Compound 7b
exhibited a twisted conformation of the two aryl groups, which
were buried in the pocket. The aniline NH group forms a
strong hydrogen bond with the backbone carbonyl oxygen of
Leu292. The 3-trifluoromethyl group interacts with a large
hydrophobic pocket formed by Tyr104, Ile299, Thr300, Ile332,
Tyr352, Ala353, and Ala356 through van der Waals
interactions. A π−π stacking interaction between the
trifluoromethylphenyl group in 7b and the Tyr104 side chain
was also observed. The lactam carbonyl group in 7b forms a
hydrogen bond with the side chain amide NH2 group of
Asn271. In addition, the lactam-fused phenyl group forms a
π−π interaction with the Tyr352 side chain.
a
b
The solubility of a series of potent KSP inhibitors was
evaluated by a thermodynamic method (Table 2).20 A mixture
of EtOH−phosphate buffer (pH 7.4) (1:1) [50% EtOH] was
Inhibition of microtubule-activated KSP ATPase activity. IC50 values
were derived from the dose−response curves generated from triplicate
data points.
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retention time. ClogP values of a series of compounds were
similar (3.9−4.2).
The solubility of a solid inhibitor in water depends on two
factors: the crystallinity of the compound and the ability of the
compound to interact with water.22 Recently, trials to address
solubility issues of drug candidates based on X-ray crystal
structures have been reported.23 Thus, we analyzed the crystal
structures of the series of inhibitors to investigate the possible
correlation with solubility (Figure 3). First, single crystals of
biphenyl-type inhibitor 4 were prepared by slow evaporation of
Et2O for X-ray structural analysis. Eight equivalent biphenyl
molecules 4 in a unit cell exhibited a slightly twisted
conformation with a relatively small dihedral angle between
the two phenyl groups (34.0°) (Figure 3A,B). Two phenyl
groups of 4 are involved in a number of CH/π interactions with
the methylene groups on lactam moieties or with the phenyl
groups of adjacent molecules, which would possibly contribute
to large van der Waals interactions for crystal packing. The
electrostatic stabilization in this packing mainly came from two
intermolecular hydrogen bonds between the lactam amide
groups across a pair of molecules. The packing energy of 4
calculated from the crystal structure by the MM method was
−55.2 kcal/mol (vdW, −37.6 kcal/mol; electrostatic, −17.6
kcal/mol) (Table 2).
Single crystals of carbazole 6a, which were obtained from an
EtOH solution,24 contained eight carbazole molecules, four
EtOH molecules, and two water molecules in a unit cell (Figure
3C,D). In the packing modes of crystals, intermolecular π−π
stacking interactions with the adjacent carbazoles were clearly
observed (average distance between two carbazole rings: 3.81
Å). The carbazole moiety also formed CH/π interactions with
other molecules of 6a, but the number of interactions was less
when compared with that of 4. Regarding electrostatic
interactions, there were two hydrogen bonds between the
lactam amide groups across a pair of molecules of 6a as
observed in the biphenyl 4 crystal. Additionally, the carbazole
NH group in 6a also interacted with an oxygen atom of EtOH
or water via a hydrogen bond, leading to an increase in the
Figure 2. Docking mode of diphenylamine 7b at the interface of
helices α4 and α6.
employed as aqueous media.20 In the solution, the parent
carbazole-type inhibitor 3 was moderately soluble (0.424 mg/
mL). Biphenyl compound 4 showed the lowest solubility in this
examination (0.339 mg/mL in 50% EtOH). Carbazoles 6a,b
were slightly more soluble (0.457 mg/mL and 0.468 mg/mL,
respectively, in 50% EtOH) compared with biphenyl 4.
Diphenylamine 7b was approximately four times more soluble
(1.80 mg/mL in 50% EtOH) than carbazoles 6a,b.21 The
physicochemical parameters such as melting point and
retention time on a reversed-phase HPLC column were also
investigated to estimate the underlying factors contributing to
the solubility. The melting point of diphenylamine 7b (190 °C)
was significantly lower than those of biphenyl 4 (221 °C) and
carbazoles 6a,b (>300 °C). In contrast, there were no
correlations between the solubility in 50% EtOH and HPLC
Table 2. KSP Inhibitory Activities and Physicochemical Properties of Biphenyl 4, Carbazole 6a,b, and Diphenylamine 7b with a
Lactam-Fused Structure
a
b
Inhibition of microtubule-activated KSP ATPase activity. IC50 values were derived from the dose−response curves generated from triplicate data
c
d
points. Solubility in 50% EtOH [an equal volume of EtOH and 1/15 M phosphate buffer (pH 7.4)]. The solubility in phosphate buffer (pH 7.4) of
compounds 4, 6a,b, and 7b was <1 μg/mL. Contributions of van der Waals (vdW) and electrostatic (ele) energies are shown in the brackets. Not
tested. CLogP values were calculated with ChemBioDraw Ultra 12.0. HPLC analysis was carried out on a Cosmosil 5C18-ARII column (4.6 × 250
e
f
g
h
mm), and the material eluted by a linear MeCN gradient (70:30 to 30:70 over 40 min) in 0.1% TFA; flow rate of 1 mL/min.
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Figure 3. X-ray crystal structures of biphenyl-type (4), carbazole-type (6a), and diphenylamine-type (7b) KSP inhibitors. (A) ORTEP diagram; (B)
crystal packing of 4. (C) ORTEP diagram; (D) crystal packing of 6a. (E) ORTEP diagram; (F) crystal packing of 7b.
electrostatic interaction contribution for the total packing
energy when compared with biphenyl 4. The average value
from four unique carbazole molecules in a unit cell was
calculated for the packing energies of 6a [−58.8 kcal/mol
(vdW, −31.8 kcal/mol; electrostatic, −27.0 kcal/mol)] (Table
2 and Supporting Information).
molecules to affect the crystal packings (Figure 3B,D,F). The
total energies of the crystal packings correlated with the order
of the melting points. For example, the higher total packing
energy may rationalize the higher melting point of carbazole 6a.
However, the thermodynamic solubility of a series of
compounds was not estimated directly from the total energy
of the crystal packing nor the melting points. The lower
solubility of 4 could be explained by the high energy of the
crystal packing from van der Waals interactions via a number of
CH/π and π−π interactions in the periodic lattice. Because
electrostatic interactions, including intermolecular hydrogen
bonds, in the crystals can be offset in aqueous solution by
interactions with the surrounding solvent, the energy from
electrostatic interactions may have less of an impact to the
solubility. In the course of the calculation of the packing
energies, the uncharged forms of three inhibitors were
compared.25 The hydrogen-bond acceptor nitrogen of the
aniline moiety in diphenylamine 7b, which is not involved in
electrostatic interactions in the crystal, may have a favorable
effect on solvation for the increased solubility. Indeed, the
solubility of 7b was much higher than that of carbazole 6a, even
though both molecules had similar packing energy values from
van der Waals interactions.
Crystal structure analysis of 7b was then performed using
colorless single crystals from EtOAc in which four equivalent
molecules were included in a unit cell (Figure 3E,F). No π−π
interactions of the aryl groups in 7b with the adjacent
molecules were involved in the crystal packing, as we expected
given that the possibly large torsion angle between the two
phenyl groups in the diphenylamine 7b would collapse the
intermolecular π−π stacking of planar carbazole 6a. Two
intermolecular CH/π interactions in a pair of molecules of 7b
between a CF3−phenyl group and a methylene C−H group of
the lactam rings were observed. The aniline NH group in 7b
formed an intermolecular hydrogen bond with a lactam
carbonyl oxygen. A pair of intermolecular hydrogen bonds
between the lactam amides as observed in biphenyl 4 and
carbazole 6a were observed. The total energy of the crystal
packing of 7b was found to be the lowest [−53.7 kcal/mol
(vdW, −29.7 kcal/mol; electrostatic, −24.0 kcal/mol)] among
three KSP inhibitors (Table 2).
Given the unique relevance of crystal packing to the
solubility, further optimization from diphenylamine 7b was
attempted for novel KSP inhibitors with more potent
bioactivity and favorable properties. Since the left-hand
lactam-fused phenyl group plays a pivotal role in KSP
Taken together, the packing modes of the crystals of the
three KSP inhibitors were revealed by X-ray crystal structure
analysis. All crystal structures share common intermolecular
hydrogen bonds across the lactam amide groups of two
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inhibition, replacement of the right-hand 3-CF3-phenyl group
with aromatic nitrogen heterocycles (8a−e) was examined
(Table 3). 5-Pyridine 8c and pyrazine derivatives 8e with a
In conclusion, we have designed a new class of KSP
inhibitors with a diaryl amine scaffold to improve aqueous
solubility from carbazole-based KSP inhibitors. The kinetic
analysis of KSP ATPase activity and molecular modeling study
suggested that diphenylamine 7b binds to the interface of α4
and α6 via van der Waals interactions and two hydrogen bonds,
working as an ATP-competitive inhibitor. The diphenylamine
7b showed four times greater solubility than the parent
carbazoles 6a,b. The investigations on the physicochemical
properties and packing energy calculations of the crystals of a
series of compounds revealed that the improvement in
solubility of 7b resulted from the reduction in van der Waals
interactions of CH/π and π−π, as well as increased solvation by
a hydrogen-bond acceptor nitrogen of the aniline moiety.
Further optimization of the right-hand CF3-phenyl group in 7b
provided a potent and more soluble KSP inhibitor 8b with a
pyridinylamine moiety.
Table 3. KSP Inhibitory Activities and Thermodynamic
Solubility of Diaryl Amines with a Right-Hand Aromatic
Heterocycle
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures, characterization, bioassay data, and
crystallographic information. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*(S.O.) Tel: +81-75-753-4551. Fax: +81-75-753-4570. E-mail:
soishi@pharm.kyoto-u.ac.jp.
*(N.F.) E-mail: nfujii@pharm.kyoto-u.ac.jp.
Funding
This work was supported by Grants-in-Aid for Scientific
Research; and Platform for Drug Discovery, Informatics, and
Structural Life Science from MEXT, Japan. T.T. is grateful for
JSPS Research Fellowships for Young Scientists.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
a
b
■
Inhibition of microtubule-activated KSP ATPase activity. IC50 values
were derived from the dose−response curves generated from triplicate
data points. Solubility in an equal volume of EtOH and 1/15 M
phosphate buffer (pH 7.4).
We thank Dr. Mikio Yamasaki and Dr. Takashi Matsumoto
(Rigaku Corporation) for their supports for X-ray analysis.
c
ABBREVIATIONS
■
nitrogen at the 5-position decreased the potency for KSP
inhibition [IC50(8c) = 0.71 μM; IC50(8e) = 1.9 μM]. 2-
Pyridine 8a and pyrimidine derivatives 8d also showed
moderate inhibitory activity [IC50(8a) = 0.41 μM; IC50(8d)
= 0.10 μM]. However, 6-pyridine 8b retained the highly potent
KSP inhibitory activity seen for the parent 7b [IC50(8b) =
0.068 μM].
KSP, kinesin spindle protein; CENP-E, centromere-associated
protein E; MKLP-1, mitotic kinesin-like protein 1
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