Synthesis and structure-activity relationship study of antimicrotubule agents phenylahistin derivatives with a didehydropiperazine-2,5-dione structure

Article abstract of DOI:10.1021/jm2009088

Plinabulin (11, NPI-2358) is a potent microtubule-targeting agent derived from the natural diketopiperazine "phenylahistin" (1) with a colchicine-like tubulin depolymerization activity. Compound 11 was recently developed as VDA and is now under phase II clinical trials as an anticancer drug. To develop more potent antimicrotubule and cytotoxic derivatives based on the didehydro-DKP skeleton, we performed further modification on the tert-butyl or phenyl groups of 11, and evaluated their cytotoxic and tubulin-binding activities. In the SAR study, we developed more potent derivatives 33 with 2,5-difluorophenyl and 50 with a benzophenone in place of the phenyl group. The anti-HuVEC activity of 33 and 50 exhibited a lowest effective concentration of 2 and 1 nM for microtubule depolymerization, respectively. The values of 33 and 50 were 5 and 10 times more potent than that of CA-4, respectively. These derivatives could be a valuable second-generation derivative with both vascular disrupting and cytotoxic activities.

Full text of DOI:10.1021/jm2009088

Article
pubs.acs.org/jmc
Synthesis and Structure−Activity Relationship Study of
Antimicrotubule Agents Phenylahistin Derivatives with a
Didehydropiperazine-2,5-dione Structure
Yuri Yamazaki,†,‡ Koji Tanaka,‡ Benjamin Nicholson,§ Gordafaried Deyanat-Yazdi,§ Barbara Potts,§
Tomoko Yoshida,‡ Akiko Oda,‡ Takayoshi Kitagawa,‡ Sumie Orikasa,‡ Yoshiaki Kiso,‡ Hiroyuki Yasui,∥
Miki Akamatsu,⊥ Takumi Chinen,# Takeo Usui,# Yuki Shinozaki,† Fumika Yakushiji,† Brian R. Miller,§
Saskia Neuteboom,§ Michael Palladino,§ Kaneo Kanoh,∇ George Kenneth Lloyd,§ and Yoshio Hayashi*,†,‡
†Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan
‡Department of Medicinal Chemistry, Center for Frontier Research in Medicinal Science, Kyoto Pharmaceutical University, Kyoto
607-8412, Japan
§Nereus Pharmaceuticals, San Diego, California 92121, United States
∥Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University, Kyoto 607-8414, Japan
⊥Laboratory of Comparative Agricultural Science, Division of Environmental Science and Technology, Graduate School of
Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
#Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
∇Marine Biotechnology Institute Co., Ltd., Kamaishi, Iwate 026-0001, Japan
S
* Supporting Information
ABSTRACT: Plinabulin (11, NPI-2358) is a potent micro-
tubule-targeting agent derived from the natural diketopiper-
azine “phenylahistin” (1) with a colchicine-like tubulin
depolymerization activity. Compound 11 was recently
developed as VDA and is now under phase II clinical trials
as an anticancer drug. To develop more potent antimicrotu-
bule and cytotoxic derivatives based on the didehydro-DKP
skeleton, we performed further modification on the tert-butyl
or phenyl groups of 11, and evaluated their cytotoxic and
tubulin-binding activities. In the SAR study, we developed
more potent derivatives 33 with 2,5-difluorophenyl and 50 with a benzophenone in place of the phenyl group. The anti-HuVEC
activity of 33 and 50 exhibited a lowest effective concentration of 2 and 1 nM for microtubule depolymerization, respectively.
The values of 33 and 50 were 5 and 10 times more potent than that of CA-4, respectively. These derivatives could be a valuable
second-generation derivative with both vascular disrupting and cytotoxic activities.
dazole13 were reported, none are as yet approved except for
colchicine as a drug for treating acute gouty arthritis.
INTRODUCTION
Antimicrotubule agents have contributed to effective chemo-
■
therapy for a variety of cancers.1 In particular, the microtubule-
Agents acting at the colchicine binding site induce a tumor-
selective vascular collapse, resulting in the prevention of blood
stabilizing taxanes2 and vinca alkaloids,3 which recognize taxoid
supply to tumor tissues, which leads to the regression of the
and vinca alkaloid sites on tubulin, respectively, are routinely
tumor.14 We have been focusing on a low molecular weight
used in the clinic. However, after long-term treatment, tumors
natural product, phenylahistin (PLH, 1, halimide), which has a
typically become resistant to these compounds.4 Therefore,
novel diketopiperazine structure (Figure 1) and acts at the
colchicine binding site on tubulin.15 This compound inhibits
there is a significant need for the development of novel
antimicrotubule agents for use in clinical oncology. Another
tumor cell growth in vitro with a submicromolar cytotoxic IC50
chemotherapeutic target site on tubulin is the colchicine
binding site, whose ligands generally inhibit tubulin polymer-
ization. Although a number of natural products and their
derivatives that recognize the colchicine site such as colchicine,5
podophyllotoxin,6 combretastatin A-4 (CA-4),7 curacin A,8
ZD6126,9 AVE8062A,10 ABT-751,11 steganacine,12 and noco-
value against a variety of tumor cell lines and shows mild
antitumor activity in the P388 leukemia and Lewis lung
carcinoma models in mice.15c
Received: July 11, 2011
Published: December 20, 2011
© 2011 American Chemical Society
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Figure 1. Structures of (−)-1, 2, 11, colchicine, and combretastatin A-4.
Compound 1 was discovered as a mixture of enantiomers
from the agar-cultured medium of Aspergillus ustus NSC-F038
by Kanoh et al.15a by the screening of new cell cycle inhibitors
and independently as halimide from a marine microorganism
by Fenical et al.16 The more potent enantiomer (−)-1
specifically inhibited the cell cycle at the G2/M phase through
the inhibition of tubulin polymerization, which is important for
spindle formation in the mitotic process. In competitive binding
assays using radiolabeled ligands, (−)-1 interacted at or near
the colchicine binding site on tubulin, but not with the vinca
alkaloid recognition site.15b
The chemical structure of (−)-1 is that of a 2,5-
diketopiperazine (DKP) derivative, consisting of L-phenyl-
alanine and a unique (Z)-isoprenylated dehydrohistidine
residue, as shown in Figure 1. This DKP provided a novel
heterocyclic template for inducing antimicrotubule activity and
a relatively hydrophilic template in comparison to that of the
other naturally occurring antimicrotubule agents mentioned
above. Therefore, the design and synthesis of highly potent
compound 1 derivatives were thought to be a valuable strategy
to develop a new anticancer drug with pharmacological,
toxicological, and resistance profiles distinct from other
antimicrotubule agents. Moreover, the simple chemical
structure of compound 1 derivatives would be manufactured
at a low price, and this would contribute to the medical
economy.
In our previous studies of chemically modified or totally
synthesized compound 1 derivatives,17 it was understood that
important structural requirements that elicit potent cytotoxic
activity were (1) the L-form of the Phe residue, (2) a rigid and
planar pseudotricyclic structure formed by hydrogen bonding
between DKP and imidazole rings, and (3) the gem-dimethyl
structure at the 5-position of the imidazole ring (Figure 2).
Kanzaki et al. reported that a biotransformed derivative of
compound 1 with an additional α,β-unsaturation joining the
DKP core with the benzyl substituent (dehydrophenylahistin,
2, Figure 1) showed a highly increased antipronuclear fusion
activity in sea urchin eggs.18 On the basis of this information,
we focused on compound 2 to develop highly potent cytotoxic
derivatives of compound 1. Namely, modifications at the
phenyl and dimethylallyl moieties were carried out to
understand the precise structure−activity relationship (SAR)
of the derivatives.
Figure 2. Structural entities required for potent biological activity of
compound 1.
cytotoxic activities were evaluated. Compound 11 showed a
further 2.8-fold increase in cytotoxic potency against human
colon adenocarcinoma cell lines (HT-29), as compared to
compound 2 (Table S1 in the Supporting Information). The
cytotoxic activity of 11 was similar to that of colchicine. Similar
results were observed in the microtubule polymerization assay
and for cytotoxic activity against HeLa cells, suggesting that the
tert-butyl group at the 5-position of the imidazole ring is
preferable for potency. In addition, compound 11 exhibited a
similar biological effect to the other antimicrotubule agents
(e.g., paclitaxel and nocodazole), as cell cycle progression of
HeLa cells was inhibited at the G2/M phase (Figure S1 in the
Supporting Information). This result suggests that the major
cytotoxic function of compound 11 is derived from the
inhibition of microtubule polymerization. The potency of
compound 11 was maintained in multidrug resistant (MDR)
tumor cell lines and was able to rapidly induce tubulin
depolymerization and monolayer permeability in normal
human umbilical vein endothelial cells (HuVECs).19a Fur-
thermore, the activity of compound 11 against human prostate
carcinoma cell line (DU 145) was further investigated in an in
vivo tumor model. A dose-dependent reduction in tumor
growth rate and tumor size was observed, with animals treated
with 3.75 or 7.5 mg/kg compound 11 having significantly
smaller tumors relative to vehicle control treated mice (Figure
S2A,B in the Supporting Information). Compound 11 was well-
tolerated, with all the mice gaining weight over the duration of
the experiment (Figure S2C in the Supporting Information).
On the basis of these and other preclinical tumor models,
tumor blood flow measurements, pharmacokinetics, distribu-
tion, metabolism, excretion, and safety testing (data not
shown), compound 11 was selected as a clinical candidate
In this SAR study, we developed its tert-butyl derivative (11,
KPU-2/NPI-2358, Figure 1),19 and its antimicrotubule and
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a
Scheme 1. Synthesis of Compounds 11−65
a
Reagents and conditions: (a) pivalic anhydride, DBU, THF, rt, 16 h, 99%; (b) formamide, reflux (165 °C), 14 h, 59%; (c) LiAlH4, THF, 0 °C to rt,
4 h, 69%; (d) MnO2, acetone, rt, 79%; (e) Cs2CO3, DMF, rt, 12 h; (f) Cs2CO3, DMF, 80 °C, 4 h.
Table 1. Cytotoxic Activity of Compound 11 Derivatives with Substitutions of 5-Position at the Imidazole
a
compd
R1
Kd (μM)
IC50 (nM)/HT-29
11
12
tert-butyl
methyl
1.0 0.5
7.4 2.3
4.9 1.4
4.6 1.1
1.1 0.3
2.0 0.7
3.3 0.3
14.9 3.8
339 41.0
153 18.0
112 16.0
15.5 1.9
30.5 4.5
16 3.0
13
n-propyl
n-butyl
14
15
isopropyl
sec-butyl
16
colchicine
a
Values are the mean or mean SEM (for only potent compounds) from at least three independent dose−response curves.
with the USAN “plinabulin” and is currently in phase II clinical
trials. In this report, we described the synthetic route of 11,
which was not disclosed, and report further derivatization from
11 to develop second-generation compounds. Then, the
dissociation constant of binding (Kd) to tubulin and the effect
on HuVECs were assessed in order to understand the vascular
disrupting ability of the derivatives.
to aldehyde, MnO2 was employed for the oxidation to prepare
aldehyde 7. The total yield of imidazolealdehyde 7 was 32%
from 3 in four steps.
In the construction of the bisdehydro-DKP template, we
modified the reported sequential two-step condensation
reaction between diacetyl-2,5-piperazinedione 8 and two
different kinds of aldehydes in the presence of Cs2CO3 as a
base in DMF.22 Namely, compound 8 was reacted with the first
aldehyde at room temperature for 12 h, followed by the
reaction with the second aldehyde at 80 °C for 4 h in degassed
DMF under an Ar atmosphere. The degassed conditions were
employed to avoid probable oxidation of activated α-carbon
atoms at the DKP ring in the presence of Cs2CO3.23
Comparison of the two synthetic routes differing in the order
of condensation of the two aldehydes indicated that aldehyde 7
should be condensed first (Scheme 1, route A) to increase the
yield of 11. The low reactivity of aldehyde 7 is attributed to the
high steric hindrance imposed by the bulky tert-butyl group at
the 5-position of the imidazole ring. After reverse-phase
CHEMISTRY
■
To synthesize various derivatives of compound 2, we examined
the effective synthetic route of 11. As shown in Scheme 1, the
synthesis of the tert-butylimidazole aldehyde 7 was commenced
with condensation of ethyl isocyanoacetate 3 with pivalic
anhydride in the presence of 1,8-diazabicyclo[5.4.0]undec-7-
ene (DBU), following the report by Suzuki et al.,20 to give
oxazole ester 4. After purification using silica gel column
chromatography, oxazole ester 4 was converted to imidazole
ester 5 by heating in formamide,21 which was reduced with
LiAlH4 in THF to give alcohol 6. In the conversion of alcohol
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Table 2. Effect of the Methoxy Group Substitution on Cytotoxic Activity
a
compd
R1
R2
R3
R4
R5
IC50 (nM)/HT-29
11
17
18
19
20
21
22
23
24
25
H
H
H
H
H
14.9 3.8
75.5 25
26.1 8.5
6570
OMe
H
H
H
H
H
OMe
H
H
H
H
H
OMe
H
H
H
OMe
OMe
OMe
OMe
H
OMe
H
H
H
868
OMe
H
H
H
5080
H
OMe
H
H
970
H
H
OMe
H
5170
OMe
OMe
OMe
H
H
2020
H
OMe
H
45.4 12
a
Values are the mean or mean SEM (for only potent compounds) from at least three independent dose−response curves.
Table 3. Cytotoxic Activity of Compound 11 Derivatives with Substitutions on the Phenyl Ring
a
compd
R1
R2
R3
R4
R5
Kd (μM)
IC50 (nM)/HT-29
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
Me
H
H
H
H
H
H
H
H
H
H
F
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
2.6 0.4
2.9 0.1
8.9 2.2
8.0 0.7
45.8 11
46.7 10
483
Me
H
H
Me
H
H
H
F
H
Me
F
H
430
b
H
nt
30.0 17
13.1 5.3
501
H
F
0.7 0.1
11.8 2.4
0.20 0.11
2.5 1.4
1.6 0.3
H
H
c
F
H
H
H
H
Cl
H
H
H
Br
H
H
NO2
H
H
H
H
H
H
H
H
2.6 0.5
46.3 13.5
24.5 8.7
709
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
b
H
H
nt
b
Cl
Br
H
Cl
nt
29.9 4.5
40.5 11
31.1 7.3
714
b
H
nt
Br
1.9 0.7
b
H
H
nt
b
NO2
H
H
nt
50.3 12.4
44.7 11.9
>20000
b
NO2
H
nt
b
H
nt
b
OH
H
H
nt
6100
b
OH
OEt
CN
vinyl
CF3
benzoyl
H
nt
364
b
H
nt
45.9 10.2
35.6 14
17.0 1.52
44.0 7.49
1.4 0.4
24.9 1.31
b
H
nt
b
H
nt
b
H
nt
c
H
0.62 0.07
b
F
nt
a
b
c
Values are the mean or mean SEM (for potent compounds) from at least three independent dose−response curves. nt = not tested. The
activity against porcine tubulin. The Kd value of compound 11 against porcine tubulin was similar (1.06 μM) to that against bovine tubulin (1.0 μM).
preparative high-performance liquid chromatography (HPLC)
purification using an octadecylsilyl (ODS) column in a dark
place, a yellow solid of 11 with >98% purity was obtained as a
trifluoroacetic acid (TFA) salt. The total yield of compound 11
via route A was 37% in two steps while the yield via route B was
only about 0.4%. Therefore, a variety of derivatives 12−65 for
the SAR study were synthesized according to route A. In the
synthesis of compounds 12−17, imidazolealdehydes with
different alkyl chains at the 5-position were also prepared
with corresponding acylanhydrides according to the same
synthetic scheme starting from isonitrile 3 (Schemes S1 and S2
in the Supporting Information). The final HPLC purification of
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every derivative removed the undesired E-forms (about 10%) at
the benzylidene, which were generated as byproducts of the
condensation reaction or upon exposure to ambient light
during the workup process. The purity of each synthesized
derivarive prior to biological evaluation was over 95%.
Derivatives with the E-configuration at the imidazolemethyli-
dene moiety were not observed, indicating that the reaction of
8 with aldehyde 7 was Z-form selective due to the hydrogen
bond formation between imidazole and DKP nitrogen atoms.
is probably different from that of colchicine, suggesting that
compound 11 recognizes tubulin in a different manner than
colchicine and its biological homologues.
An expanded SAR study on the phenyl ring was performed
by means of the introduction of a variety of substituents. As
shown in Table 3, in the case of single substitutions with the
methyl group, halogen atoms or a nitro group, ortho- or meta-
substituted derivatives exhibited relatively high potency in both
tubulin binding and cytotoxic assays, while all efforts at the
para-position completely failed to exhibit even moderate
potency. Among the ortho- or meta-substituted derivatives,
compound 31 with the m-fluoro group showed potent cytotoxic
activity (IC50 = 13.1 nM), which was similar to that of
compound 11. Introduction of a hydroxyl group at the ortho-
or meta-position resulted in significant reduction of potency.
The m-hydroxyl derivative 45 exhibited a 16-fold higher
potency for cytotoxic activity than the o-hydroxyl derivative
44, but was 24-fold less potent than compound 11. The
attenuated activity of the m-hydroxyl derivative suggests that a
hydrogen bond donor at this position is undesirable; however, a
hydrogen bond acceptor at this position may be tolerated, since
the m-fluoro analog maintained good potency. The comparable
activity of the m-fluoro analog with 11 may simply reflect the
isosteric nature of the fluoro group. Overall, while single
substitutions of the phenyl ring did not improve potency
relative to 11, substitutions at the meta-position were generally
well-tolerated, suggesting an opportunity for further explora-
tion.
RESULTS AND DISCUSSION
■
SAR Study from Compound 11. 1. Modification of the
Alkyl Group on the Imidazole Ring. To perform the
optimization on the 5-position of the imidaole ring, the tert-
butyl group of compound 11 was replaced to other alkyl groups
and the synthesized derivatives were evaluated in both the
cytotoxic and tubulin-binding assays (Table 1). The derivatives
with normal alkyl groups, i.e., methyl (compound 12), n-propyl
(compound 13), and n-butyl (compound 14) groups, showed
significantly lower potency than compound 11. However, the
derivatives with blanched alkyl groups, i.e., isopropyl (com-
pound 15) and sec-butyl (compound 16) groups maintained
potency, particularly isopropyl derivative, which exhibited the
same potency as compound 11. This result suggests that the
blanched chain at 5-position of the imidazole ring is critical to
form a tight binding with tubulin and to exhibit a potent
cytotoxic activity and is consistent with earlier findings on
(−)-1.17a
For the double modifications at the phenyl ring, 2,5-difluoro
subsutitution (compound 33) showed an increase in potency of
5.7-fold (IC50 = 2.6 nM) compared to compound 11. This may
reflect the unique properties of fluorinated organic compounds,
including improved entropies of ligand binding associated with
these agents.25 On the other hand, 2,3-dichloro (compound
37) and 2-fluoro-6-chloro (compound 51) substitutions
resulted in only slight decreases in potency, while the 2,6-
dimethyl substitution (compound 29) resulted in low potency.
These results supported further modifications to the meta-
position and indicated that the phenyl ring is specifically
recognized by tubulin in a highly restricted manner.
Since our examination indicated that modification at the
meta-position generally resulted in a greater potency than that
of the ortho-position, further modifications to the meta-
position were undertaken to increase potency. A vinyl group
substitution at the meta-position (compound 48) showed
potent activity (IC50 = 17 nM), equivalent to that of compound
11, while substitution with the m-ethoxy (compound 46), m-
cyano (compound 47), or m-trifluoromethyl (compound 49)
group slightly decreased the potency; of note, the latter is both
isosteric and comparable in activity to the m-methyl analog 27.
With the introduction of the carbonyl group, compound 50
with a m-benzoyl group provided the most potent activity in
the present study, with 11-fold more potent cytotoxicity (IC50 =
1.4 nM) and 1.6-fold higher tubulin binding constant (Kd = 0.6
μM) than compound 11, although the derivative with a
hydrophilic m-carbamoyl group showed very weak potency
(data not shown). The enhanced potency of benzophenone
analog 50 may reflect, in part, an entropic effect, with improved
binding energetics of this more rigid ligand for its target.
Spacially, these findings suggest the existence of a narrow, but
deep, crevice in the binding site on tubulin that accepts the
additional planar structure at the meta-position. Ideally, this
crevice could effectively function as an adaptive space for
2. Modification of the Phenyl Ring. To develop more
potent antimicrotubule and cytotoxic derivatives based on
compound 11, a precise SAR study focused on its phenyl ring
was performed. It is known that natural antimicrotubule agents
such as colchicine, combretastatin, and derivatives, which
recognize the same colchicine binding site on β-tubulin as
compound 1, are structurally characterized by multiple methoxy
groups, which make marked contributions to their potent
antimicrotubule activity.24 Therefore, we evaluated analogs of
compound 11 bearing methoxy substitutuents to the phenyl
ring for cytotoxicicity to HT-29 cells. As shown in Table 2, in
the case of a single substitution, the ortho-substitution
(compound 17) resulted in a 5-fold decrease in cytotoxic
potency, while meta-substitution (compound 18) maintained
potency similar to that of compound 11. However, the para-
substitution (compound 19) resulted in a marked decrease in
potency (>400-fold). Double substitution on the phenyl ring
resulted in a marked decrease in potency for most derivatives,
except for the 3,5-substitution (compound 25), which showed a
slightly weaker potency than compound 11. These results
indicate that both meta-positions have a potencial for the
modification of potency. Methoxy derivatives with triple
substitutions similar to those occurring in colchicine demon-
strated low potency (data not shown). These results suggest
that tubulin recognizes the phenyl ring of compound 11
rigorously and the space for accepting the para-substituent is
highly limited. In addition, in comparison to the relatively high
potency of the single ortho-substitution (compound 17), the
observed reduction in potency of the double ortho-substitution
(compound 23) suggests that proper spatial orientation of the
phenyl ring is critical for the activity. Specifically, rotation of the
ring induced by steric hindrance may preclude the derivatives
from adopting the most active conformation. Moreover, the
collective SAR data for this series suggest that the recognition
of the methoxy groups by the colchicine binding site on tubulin
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increasing the activity of compound 50. Finally, the hydro-
phobic property of the benzophenone moiety of compound 50
might increase cell penetration, leading to highly potent
cytotoxic activity in comparison to tubulin binding potency.
Encouraged by the results of replacing the phenyl ring with
benzophenone, we expanded the SAR study to explore
replacement with other ring systems; the results on tubulin
binding and cytotoxic assays are shown in Table 4.
of a basic nitrogen into compound 1 derivatives provides added
value.
The pyridine derivatives 56−58 showed decreased potency,
although each position of the nitrogen atom on the pyridine
ring afforded a different influence. As described above, the
meta-position was relatively well tolerated. However, the 2-
pyridyl derivative 56 was completely inactive. Since the 2-
pyridyl nitrogen atom could form a hydrogen bond with the
DKP amide NH via a pseudo-six-membered-ring formation, the
conformation of the pyridine ring would be able to be fixed on
the same plane as the DKP ring, resulting in a planar
pseudopentacyclic conformation. This suggested that a certain
degree of spatially distorted positioning between both rings was
critical for the activity (Figure 4).
Table 4. Cytotoxic Activity of Compound 11 Derivatives
with Other Ring Systems
We also replaced the phenyl ring with smaller ring systems
such as furan, thiophene, and pyrrole. Compound 59 with a 2-
furyl group had a very weak potency; however, the introduction
of a methyl group (compound 60) or chlorine atom
(compound 61) at the 5-position of the furan ring restored
the potency. The derivatives with 3-fulyl (compound 62), 2-
thienyl (compound 63), and 3-thienyl (compound 64) groups
also showed a slightly lower potency than compound 11. The
replacement of the phenyl ring by the cyclohexyl ring
(compound 65) resulted in a lower potency, suggesting that
the more rigid, planar aromatic ring system was favorable to
elicite the high potency in the cytotoxicity assay.
Inhibition of Microtubule Polymerization with the
Potent Derivatives. Since 2,5-difluoro derivative 33 and
benzophenone derivative 50 showed a higher potency in
cytotoxic activity against HT-29 cell lines than compound 11,
we performed in vitro microtubule depolymerization assay
using purified porcine tubulin to investigate whether these
derivatives exhibit antimicrotubule activity (Table 5). The
Table 5. Inhibition of in Vitro Tubulin Polymerization and
Tubulin Binding Activity
inhibition of tubutin polymerization tubulin binding Kd
a
compd
IC50 (μM)
(μM)
11
33
50
55
1.31 0.10
0.51 0.02
0.76 0.14
0.55 0.04
0.55 0.01
1.0 0.5
0.20 0.11
0.62 0.07
1.2 0.3
b
combretastatin
A-4
nt
a
Values are the mean or mean SEM (for potent compounds) from
b
at least three independent dose−response curves. n.t. = not tested.
colchicine
4.12 0.63
3.3 0.3
a
Values are the mean or mean SEM (for potent compounds) from
Replacement with the naphthalene ring, 1-naphthyl derivative
52, showed almost the same potency as compound 11, while
the 2-naphthyl derivative 53 exhibited very weak potency. This
observation was consistent with the result obtained with the
benzophenone derivative 50. An additional planar ring system
connected at ortho- and meta-positions of the phenyl ring was
well-tolerated for tubulin binding. The 5-isoquinoline derivative
55 showed similar potency to compound 11, although the 8-
quinoline derivative 54 was 7-fold less potent than compound
55. An appropriate alignment of the electron-donating aromatic
nitrogen is apparently important for maintaining activity.
Importantly, this basic nitrogen atom may increase the water
solubility of the derivatives as an acid salt; although the series of
compound 1 may appear to be equipped with inherent
ionization potential via the imidazole ring, conjugation of the
imidazole with the DKP core renders this imidazole an
extremely weak base (data not shown); thus, the incorporation
b
at least three independent dose−response curves. nt = not tested.
activity of the 5-isoquinoline derivative 55 was also evaluated
because it possessed favorable solubility in water (data not
shown) as well as exhibiting a similar cytotoxic activity to
compound 11. In this assay, compounds 33, 50, and 55
exhibited more potent mictotubule depolymerization activity
than compound 11 and similar activity to combretastatin A-4.
Cell Cycle Progression Analysis. The effects of
compounds 11, 33, 50, and 55 on cell cycle progression were
investigated using flow cytometry. After treatment with
different concentrations of compound for 18 h, HeLa cells
were fixed, stained with propidium iodide, and counted (Table
6 and Figures S3−S8 in the Supporting Information). All of
these derivatives caused significant arrest of the cells at the G2/
M phase, compared to the untreated control. The ability of
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4 (10 nM), respectively. The results indicated that compound
50 was the most potent VDA in the present study.
Table 6. Effect of 11, 33, 50, and 55 on Cell Cycle
Progression
Correlation between Kd and IC50 of the Derivatives.
To understand whether the cytotoxic effect of the present DKP
derivatives is dependent on their antimicrotubule activity, we
examined the relationship between the Kd (1/Ka) in the tubulin
binding assay and IC50 values in the HT-29 assay based on the
results from 19 derivatives for which both tubulin binding and
cytotoxicity assays were performed.26 As shown in Figure 3, the
cell population (%)
compd
conc (nM)
G0/G1
S
G2/M
control
0
10
30
100
3
43.9
44.6
4.2
39.9
37.5
40.4
2.4
16.2
17.9
55.3
97.6
16.4
92.3
45.7
93.0
14.2
85.5
37.4
90.7
21.0
89.9
11
0.0
33
41.5
1.2
42.1
6.7
10
3
50
55
24.7
0.5
29.6
6.5
10
30
100
3
48.2
9.8
37.6
4.8
combretastatin A-4
colchicine
34.6
0.4
28.1
8.8
10
10
30
47.5
1.3
31.5
8.9
compound 33 to induce arrest of the cell cycle was more potent
than that of 11, although compound 55 showed less potency
than 11. Compound 50 caused maximam arrest of the cell at
the G2/M phase at the concentration of 3 nM, and its potency
was similar to that of combretastatin A-4. The order of cell
arrest ability at the G2/M phase was 50 > 33 > 11 > 55,
consistent with their cytotoxic activities against HT-29 cells.
In Vitro Vascular Disrupting Activity. Compound 11
significantly increased endothelial cell permeability, and this
activity was more potent than that of either colchicine or
vincristine, while the profile of combretastatin A-4 was similar
to that of compound 11.19a In addition, compound 11 rapidly
induced morphological changes such as membrane blebbing
and the rounding up of cells in HuVECs, which is propbably a
downstream effect of 11-mediated microtubule disruption. To
understand the vascular disrupting function of the present
plinabulin derivatives, cytotoxic and antimicrotubule activities
of compounds 33, 50, and 55 against HuVECs were examined.
As shown in Table 7, all of the compound 2 derivatives
Figure 3. Correlation of the binding activity to purified porcine
tubulin (Ka) vs cytotoxic activity (IC50) among 19 compounds: (filled
circle) compounds 11−16, 26−29, 31−35, 39, 54, and 55; (open
circle) compound 50.
double reciprocal plots of each value from 18 compounds,
except for compound 50, showed a high positive correlation
(R2 = 0.993), suggesting that derivatives of 11 have a high
correlation between tubulin binding and cytotoxic potency.
Therefore, it is revealed that the cytotoxic activity of the DKP
derivatives is largely attributed to their antimicrotubule action.
On the other hand, compound 50 with the benzophenone
structure appeared to fall outside of this correlation, with high
cytotoxic potency in comparison to its tubulin binding.
Considering the result of tubulin depolymerization assay and
cell cycle analysis, it is suggested that the benzophenone
derivative 50 might have the another mode of action, such as a
higher cell penetration property, in addition to the tubulin
depolymerization in order to induce the potent cytotoxicity.
Active Conformation of the Derivatives. Conforma-
tional analysis of the compound 1 derivatives may provide
insights into the active conformation that supports ligand
binding to tubulin. Clearly, the conformation of compound 1
derivatives is tightly restricted by the pseudotricyclic structure
formed by the hydrogen bonding between the imidazole and
DKP rings, as supported by the crystal structure of 11.
However, conformational analysis of the rotatable phenyl ring
has not been previously explored. Thus, the X-ray crystal
structures of potent compound 11 and inactive compound 56,
with the o-pyridyl group, were analyzed. As shown in Figure 4B,
inactive compound 56 formed an additional hydrogen bond
between the o-pyridine nitrogen atom and the NH group of the
DKP ring, which was apparent in the 1H NMR spectra,
resulting in the planar structure. This result indicated that the
uniplanar structure with a pseudotricyclic ring represented a
Table 7. Antimicrotubule and Cytotoxic Activities of
Compound 11 Derivatives against HuVECs
30 min HuVECs microtubule
depolymerization
IC50 (nM)
a
ED100
doses tested
(nM)
b
compd
/HuVECs
(nM)
11
11 0.6
1.5 0.3
0.5 0.1
3
2
3, 5, 10
33
1, 2, 5, 50
0.1, 1, 5, 10
10, 20, 50
2, 10, 20, 50
50
55
1
13
4
20
10
combretastatin A-4
2.4 0.8
a
Values are the mean or mean SEM (for potent compounds) from
b
at least three independent dose−response curves. Lowest concen-
tration tested where complete depolymerization was observed.
demonstrated slightly greater potency against HuVECs than
HT-29 cells. Particularly, compound 50 showed 22-times
greater potency than compound 11, with an IC50 value of 0.5
nM, which was about 5-times more potent than combretastatin
A-4. Moreover, the lowest effective dose of compound 50 for
antimicrotubule activities was 1 nM, which was 3 and 10 times
more potent than compound 11 (3 nM) and combretastatin A-
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CONCLUSION
■
A series of highly potent and low molecular weight (<450)
antimicrotubule agents with a DKP core structure was designed
and synthesized on the basis of the modification of compound
11 derived from compound 1. SAR studies were performed on
the basis of substituents of both the benzyl and imidazole
moieties, which are protruding from the biologically important
pseudotricyclic structure of compound 1. Several synthesized
compounds inhibited tumor cell growth in vitro with
nanomolar IC50 values against HT-29 human carcinoma cells
and exhibited potent tubulin binding with a Kd value similar to
or lower than that of colchicine. In addition, compounds 33
and 50, which were more potent compounds than compound
11, also exhibited highly potent antimicrotubule and cytotoxic
activities against HuVECs, a model cell system for evaluating
vascular disrupting activity in vitro. As second-generation
compounds of 11, potent compounds 33 and 50 could
contribute toward tumor vascular disrupting therapy both as
single agent and in combination with other chemotherapeutic
agents or radiation for the treatment of solid tumors and
toward gaining a better understanding for the development of
new antimicrotubule agents for use in clinical oncology.
EXPERIMENTAL SECTION
■
Figure 4. ORTEP drawing of compounds 11 and 56. The arrow
indicates the steric repulsion between H1 and H19.
General Methods. Reagents and solvents were obtained from
Wako Pure Chemical Ind., Ltd. (Osaka, Japan), Nacalai Tesque
(Kyoto, Japan), and Aldrich Chemical Co. Inc. (Milwaukee, WI) and
used without further purification. Column chromatography was
performed on Merck 107734 silica gel 60 (70−230 mesh). TLC was
performed using Merck silica gel 60F254 precoated plates. Melting
points were measured on a Yanagimoto micromelting apparatus
without correction. Analytical HPLC was performed using a C18
reverse phase column (4.6 × 150 mm; YMC Pack ODS AM302) with
a binary solvent system: linear gradient of CH3CN in 0.1% aqueous
TFA at a flow rate of 0.9 mL/min, detected at UV 230 nm. Preparative
HPLC was carried out on a C18 reverse phase column (20 × 250 mm;
YMC Pack ODS-AM or 19 × 150 mm; Waters μBondasphere C18, 5
μm, 100 Å) with a binary solvent system: linear gradient of CH3CN in
0.1% aqueous TFA or methanol in water at a flow rate of 5−12 mL/
min, detected at UV 230 nm. Solvents used for HPLC were of HPLC
biologically unfavorable conformation. On the other hand, the
active compound 11 exhibited a twisted conformation between
the phenyl and DKP rings induced by steric repulsion (Figure
4A (arrow)). The dihedral angle was about 30°. These findings
suggest that the active conformation required a certain extent of
dihedral angle around the C13−C14 bond. This is supported
by evaluation of other analogs in the series. For example, the
low potency of 2-furyl derivative 59 may be explained by the
hydrogen bond formation between the oxygen atom on the 2-
furyl ring and the NH group of the DKP ring, as observed in
the o-pyridine derivative 56. In addition, introduction of bulky
substituents at the adjacent 5-position of the 2-furyl ring
(compound 60 and 61) might prevent this hydrogen bonding
by steric hindrance, resulting in an increase in cytotoxic
potency. The potent activity observed in the 2-thienyl
derivative 63 might be due to the presence of the sulfur
atom, which will not serve as a hydrogen bond acceptor for the
DKP NH. It is known that the active conformation of
colchicines requires the defined dihedral angles between A
and C rings,27 which is preserved among other colchicine site
binding agents.28 However, this conformational property
appears not to apply to our derivatives, because the
superimposition study between colchicine and compound 11
failed due to their low structural similarity, suggesting a
different binding mode of our derivatives at or near to the
colchicine site on tubulin.29
1
grade. All other chemicals were of analytical grade or better. H and
13C NMR spectra were obtained on JEOL 300 MHz spectrometer, a
Varian Mercury 300 spectrometer (300 MHz), Bruker DPX-400
spectrometer (400 MHz) or a Bruker AV600 spectrometer (600
MHz) with TMS as an internal standard. IR spectra were recorded on
JASCO FT/IR 4100 spectrometer. High-resolution mass spectra (ESI
or EI) were recorded on a micramass Q-Tof Ulitima API or a JEOL
JMS-GCmate BU-20 spectrometer. X-ray crystallographic analysis was
carried out on a Mac Science DIP-2020. The elemental analyses were
performed on a PerkinElmer 2400 Series II analyzer (N241-0610) or
an Elementar Vario EL elemental analyzer. The purity of all tested
compounds was measured by a reverse-phase HPLC with the
following conditions, and the purity of all compounds were >95%.
HPLC Conditions. Method 1: solvent A, 0.1% TFA−water; solvent
B, 100% acetonitrile; flow rate of 0.9 mL/min, from 0% B to 100% B
in 40 min. Method 2: solvent A, 0.1% TFA−water; solvent B, 100%
acetonitrile; flow rate of 0.9 mL/min, from 0% B to 100% B in 35 min.
Method 3: solvent A, 0.1% TFA−water; solvent B, 100% acetonitrile;
flow rate of 0.9 mL/min, from 10% B to 50% B in 40 min.
Ethyl 5-(tert-butyl)oxazole-4-carboxylate (4) [CAS No.
714273−89−9]. According to our previous report,29 to a solution
of ethyl isocyanoacetate 3 (25 g, 221 mmol) in THF (200 mL) were
added DBU (34.3 mL, 243 mmol) and pivalic anhydride (49.3 mL,
243 mmol), and the mixture was stirred overnight at room
temperature. After the solvent was removed by evaporation in
vacuo, the residue action was extracted with AcOEt (200 mL);
washed with 10% Na2CO3, 10% citric acid, and saturated NaCl three
Furthermore, 3D-QSAR was carried out using comparative
molecular field analysis (CoMFA)30 to create the pharmaco-
phore model at the phenyl moiety in 11 derivatives, since the
11-binding site on tubulin has not precisely been determined
(Figure S9 in the Supporting Information). In this analysis, the
meta-position of the phenyl ring was indicated as a favorable
area, while the para-position was indicated as an unfavorable
area for introducing the substituting group. These results are
also consistent with the findings of the SAR study, especially
concerning derivatives 31, 33, 35, and 39.
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times; and dried over anhydrous Na2SO4, and the solvent was
concentrated in vacuo. The residual oil was purified by silica gel
column chromatography using hexane−AcOEt (20:1 to 4:1) to give an
flushing process was repeated again. The resultant mixture was heated
for 4 h at 80 °C. After the solvent was removed by evaporation, the
residue was dissolved in EtOAc, washed with water two times and
saturated NaCl three times, dried over Na2SO4, and concentrated in
vacuo. The resulting residue was dissolved in 70% aqueous MeCN,
applied to a reverse-phase HPLC column (μBondasphere 5C18 100 A,
19 × 150 mm), and eluted using a linear gradient from 20 to 80%
CH3CN in 0.1% aqueous TFA over 30 min at a flow rate of 12 mL/
min, and the desired fraction was collected, concentrated by
evaporation, and then lyophilized to give 94.0 mg of the title
compound 11 as a yellow solid: yield 81%; mp 160−162 °C (dec); IR
(KBr, cm−1) 3500, 3459, 3390, 3117, 3078, 2963, 2904, 1673, 1636,
1
oil of 4 (66.4 g, 99%): H NMR (300 MHz, CDCl3) δ 7.70 (s, 1H),
4.39 (q, J = 7.2 Hz, 2H), 1.46 (s, 9H), 1.41 (t, J = 7.2 Hz, 3H); HRMS
(EI) m/z 197.1050 (M+) (calcd for C10H15NO3 197.1052).
Ethyl 5-(tert-butyl)imidazole-4-carboxylate (5). A mixture of 4
(11 g, 55.77 mmol) and formamide (66 mL, 1.66 mol) was heated at
165 °C for 14 h. After cooling, the reaction mixture was extracted with
AcOEt (200 mL) and the organic layer was washed with 10% Na2CO3
and saturated NaCl three times, dried over anhydrous Na2SO4, and
concentrated in vacuo. The residual dark brown oil was purified by
column chromatography on silica gel using CHCl3−MeOH (100:1 to
1
1601, 1413, 1371, 1345; H NMR (300 MHz, DMSO-d6) δ 12.26 (s,
1
50:1) as an eluant to give 6.44 g (59%) of a white solid 5: H NMR
2H), 10.16 (br s, 1H), 7.86 (s, 1H), 7.53 (d, J = 7.4 Hz, 2H), 7.42 (t, J
= 7.5 Hz 2H), 7.32 (t, J = 7.4 Hz, 1H), 6.86 (s, 1H), 6.75 (s, 1H), 1.38
(s, 9H); 13C NMR (150 MHz, DMSO-d6) 157.2, 156.4, 145.3, 137.4,
134.5, 133.1, 129.1, 128.6, 127.9, 126.4, 113.9, 112.0, 104.5, 37.4, 27.7;
HRMS (EI) m/z 336.1591 (M+) (calcd for C19H20N4O2 336.1586).
Anal. (C19H20N4O2·0.25H2O·CF3COOH) C, H, N. HPLC (method
1) 99.4% (tR = 18.87 min).
Compounds 12−16 were prepared from compound 8 over two
steps according to the procedure (route A) described for the synthesis
of 11.
(3Z,6Z)-3-Benzylidene-6-((5-methyl-1H-imidazol-4-yl)-
methylene)piperazine-2,5-dione (12). Yield of 3% from 8 (two
steps); mp >280 °C; IR (KBr, cm−1) 3185, 3107, 3032, 2872, 2776,
2681, 1682, 1645, 1393; 1H NMR (300 MHz, DMSO-d6) δ 12.72 (br
s, 1H), 11.85 (br s, 1H), 10.08 (s, 1H), 8.00 (s, 1H), 7.54 (d, J = 7.7
Hz, 2H), 7.42 (t, J = 7.5 Hz, 2H), 7.33 (t, J = 7.0 Hz, 1H), 6.76 (s,
1H), 6.58 (s, 1H), 2.31 (s, 3H); 13C NMR (150 MHz, DMSO-d6)
157.8, 157.0, 135.4, 133.6, 129.7, 129.5, 129.2, 128.5, 127.0, 114.5,
103.8, 9.6; HRMS (ESI) m/z 295.1201 (M + H)+ (calcd for
C16H15N4O2 295.1195); HPLC (method 1) 97.7% (tR = 12.10 min).
Anal. (C16H14N4 O2·CF3COOH·0.67H2O) C, H, N.
(3Z,6Z)-3-Benzylidene-6-((5-propyl-1H-imidazol-4-yl)-
methylene)piperazine-2,5-dione (13). Yield of 9% from 8 (two
steps); mp >280 °C (dec); IR (KBr, cm−1) 3219, 3176, 3088, 3036,
2966, 2930, 2871, 1684, 1638, 1394; 1H NMR (300 MHz, DMSO-d6)
δ 12.70 (br s, 1H), 11.91 (s, 1H), 10.09 (s, 1H), 8.00 (s, 1H), 7.54 (d,
J = 7.5 Hz, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.2 Hz, 1H), 6.76
(s, 1H), 6.58 (s, 1H), 2.68 (t, J = 7.3 Hz, 2H), 1.63−1.55 (m, 2H),
0.88 (t, J = 7.3 Hz, 3H); 13C NMR (150 MHz, DMSO-d6) 157.8,
156.9, 135.6, 133.7, 133.6, 129.7, 129.2, 128.5, 127.0, 114.5, 103.7,
25.8, 23.1, 13.9; HRMS (ESI) m/z 323.1519 (M + H)+ (calcd for
C18H19N4O2 323.1508); HPLC (method 1) 98.1% (tR = 14.76 min).
Anal. (C18H18N4O2·0.65CF3COOH·0.33H2O) C, H, N.
(300 MHz, CDCl3) δ 7.51 (s, 1H), 4.36 (q, J = 6.9 Hz, 2H), 1.46 (s,
9H), 1.40 (t, J = 7.2 Hz, 3H); HRMS (EI) m/z 196.1213 (M+) (calcd
for C10H16N2O2 196.1212).
(5-(tert-Butyl)imidazol-4-yl)methanol (6). To a solution of 5
(10 g, 50.95 mmol) in THF (200 mL) was added LiAlH4 (1.93 g,
50.95 mmol) portionwise under argon atmosphere at 0 °C, and the
mixture was stirred for 4 h at room temperature. To this solution was
added saturated aqueous NH4Cl slowly at 0 °C, and the resulting
precipitate was romoved by Celite filtration. The filtrate was extracted
with AcOEt, washed with 10% Na2CO3 and saturated NaCl twice,
dried over anhydrous Na2SO4, and concentrated in vacuo. The residual
oil was purified by silica gel chromatography using CHCl3−MeOH
(200:1) to give an oil of 6 (5.4 g, 69%): 1H NMR (300 MHz, CDCl3)
δ 7.55 (br s, 1H), 4.75 (s, 2H), 1.37 (s, 9H); HRMS (EI) m/z
154.1118 (M+) (calcd for C8H14N2O 154.1103).
5-(tert-Butyl)imidazole-4-carboxaldehyde (7). To a solution of
6 (4.1 g, 26.6 mmol) in acetone (70 mL) was added MnO2 (16.2 g,
186.2 mmol), and the mixture was stirred at room temperature for 2 h.
After filtration to remove MnO2, the solvent was removed by
evaporation, and the residual white powder was purified by silica gel
column chromatograpy using CHCl3−MeOH (200:1) as a eluate to
give 3.2 g (79%) of off-white solid 7: 1H NMR (300 MHz, CDCl3) δ
10.09 (s, 1H), 7.78 (s, 1H), 1.50 (s, 9H); HRMS (EI) m/z 152.0950
for (M+) (calcd 152.0947 for C8H12N2O).
1-Acetyl-3-{(Z)-1-[5-(tert-butyl)-1H-4-imidazolyl]-
methylidene}]-2,5-piperazinedione (9). To a solution of 5-(tert-
butyl)imidazole-4-carboxaldehyde 7 (200 mg, 1.31 mmol) in DMF (2
mL) was added compound 8 (390.7 mg, 1.97 mmol) and the solution
was repeatedly evacuated in a short time to remove oxygen and flushed
with Ar, followed by the addition of Cs2CO3 (642.6 mg, 1.97 mmol),
and the evacuation−flushing process was repeated again. The resultant
mixture was stirred for 12 h at room temperature. After the solvent was
removed by evaporation, the residue was dissolved in the mixture of
AcOEt and water, and the organic phase was washed with saturated
NaCl twice, dried over Na2SO4, and concentrated in vacuo. The
residual oil was purified by column chromatography on silica gel using
CHCl3−MeOH (200:1 to 50:1) as an eluant to give 380 mg (46%) of
a pale yellow solid 9: 1H NMR (300 MHz, CDCl3) δ 12.14 (br s, 1H),
9.22 (br s, 1H), 7.57 (s, 1H), 7.18 (s, 1H), 4.47 (s, 2H), 2.65 (s, 3H),
1.47 (s, 9H); HRMS (EI) m/z 290.1382 (M+) (calcd for C14H18N4O3
290.1379).
(3Z,6Z)-3-Benzylidene-6-((5-butyl-1H-imidazol-4-yl)-
methylene)piperazine-2,5-dione (14). Yield of 13% from 8 (two
steps); mp >270 °C (dec); IR (KBr, cm−1) 3180, 3120, 3069, 2955,
1
2928, 2870, 1675, 1635, 1418; H NMR (300 MHz, DMSO-d6) δ
12.56 (s, 1H), 12.01 (s, 1H), 10.04 (s, 1H), 7.90 (s, 1H), 7.54 (d, J =
7.5 Hz, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.2 Hz, 1H), 6.75 (s,
1H), 6.58 (s, 1H), 2.71 (t, J = 7.4 Hz, 2H), 1.60−1.50 (m, 2H), 1.35−
1.23 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H); 13C NMR (150 MHz, DMSO-
d6) 157.9, 156.7, 135.7, 134.0, 133.7, 132.8, 129.7, 129.2, 128.5, 127.1,
124.2, 114.3, 104.4, 32.0, 23.6, 22.1, 14.1; HRMS (ESI) m/z 337.1659
(M + H)+ (calcd for C19H21N4O2 337.1665); HPLC (method 1)
97.3% (tR = 16.39 min). Anal. (C19H20N4O2·0.25H2O) C, H, N.
(3Z,6Z)-3-Benzylidene-6-((5-isopropyl-1H-imidazol-4-yl)-
methylene)piperazine-2,5-dione (15). Yield of 6% from 8 (two
steps); mp >270 °C (dec); IR (KBr, cm−1) 3137, 3056, 2972, 2937,
2874, 1675,1649, 1389; 1H NMR (300 MHz, DMSO-d6) δ 12.72 (br s,
1H), 11.92 (s, 1H), 10.08 (s, 1H), 8.00 (s, 1H), 7.54 (d, J = 7.5 Hz,
2H), 7.42 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.5 Hz, 1H), 6.76 (s, 1H),
6.61 (s, 1H), 3.29−3.20 (m, 1H), 1.24 (d, J = 6.9 Hz, 6H); 13C NMR
(150 MHz, DMSO-d6) 157.8, 156.8, 139.4, 135.8, 133.7, 129.7, 129.2,
128.5, 127.0, 114.4, 110.0, 104.1, 24.5, 23.0; HRMS (ESI) m/z
323.1502 (M + H)+ (calcd for C18H19N4O2 323.1508); HPLC
1-Acetyl-3-(Z)-phenylmethylidene-2,5-piperazinedione (10)
[CAS No. 30166−30−4]. This known compound22b was synthesized
using a procedure similar to that of compound 9. Namely,
benzaldehyde (2.7 mL, 26.5 mmol) and compound 8 (5 g, 25.2
mmol) were reacted in DMF (70 mL) in the presence of Cs2CO3 (8.6
g, 26.5 mmol) under Ar atmosphere for 2 h at room temperature: yield
1
76%; H NMR (300 MHz, CDCl3) δ 7.92 (br s, 1H), 7.37−7.50 (m,
5H), 7.26 (s, 1H), 7.19 (s, 1H), 4.52 (s, 2H), 2.66 (s, 3H); HRMS
(ESI) m/z 245.0921 (M + H+) (calcd for C13H13N2O3 245.0926).
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-phenylmethylidene]-2,5-piperazinedione (11). To a solution
of 9 (100 mg, 0.34 mmol) in DMF (4 mL) was added benzaldehyde
(52.25 μL, 0.51 mmol) and the solution was repeatedly evacuated in a
short time to remove oxygen and flushed with Ar, followed by the
addition of Cs2CO3 (224.6 mg, 0.68 mmol), and the evacuation−
( m e t h o d 1 ) 9 9 . 2 % ( t R
=
1 4 . 3 1 m i n ) . A n a l .
(C18H18N4O2·CF3COOH·0.75H2O) C, H, N.
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(3Z,6Z)-3-Benzylidene-6-((5-sec-butyl-1H-imidazol-4-yl)-
methylene)piperazine-2,5-dione (16). Yield of 20% from 8 (two
steps); mp 226−228 °C; IR (KBr, cm−1) 3433, 3185, 3161, 2961,
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(2,5-dimethoxyphenyl)methylidene]-2,5-piperazinedione
(22). Yield of 5% from 9; mp 257−259 °C (dec); IR (KBr, cm−1)
1
1
3196, 3140, 2968, 1657, 1395; H NMR (300 MHz, CDCl3) δ 12.36
2876, 2679, 1692, 1670, 1399; H NMR (300 MHz, DMSO-d6) δ
(s, 1H), 9.57 (br s, 1H), 8.81 (s, 1H), 7.57 (s, 1H), 7.01 (s, 1H), 6.92
(s, 1H) 6.82−6.97 (m, 3H), 3.89 (s, 3H), 3.79 (s, 3H) 1.42 (s, 9H);
13C NMR (100 MHz, DMSO-d6) 157.1, 156.9, 153.1, 151.3, 140.2,
134.4, 126.8, 122.6, 115.8, 115.0, 112.7, 110.8, 104.0, 56.2, 55.6, 32.0,
30.5; HRMS (EI) m/z 396.1799 (M+) (calcd for C21H24N4O4
396.1797); HPLC (method 1) 98.1% (tR = 20.04 min). Anal.
(C21H24N4O4·CF3COOH) C, H, N.
12.67 (br s, 1H), 11.93 (s, 1H), 10.08 (s, 1H), 8.02 (s, 1H), 7.54 (d, J
= 7.5 Hz, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.2 Hz, 1H), 6.78
(s, 1H), 6.59 (s, 1H), 3.00−2.93 (m, 1H), 1.67−1.50 (m, 2H), 1.23 (d,
J = 7.0 Hz, 3H), 0.75 (t, J = 7.3 Hz, 3H); 13C NMR (150 MHz,
DMSO-d6) 157.2, 156.3, 137.4, 135.3, 133.1, 129.2, 128.6, 127.9,
126.4, 113.9, 103.2, 30.9, 29.2, 20.2, 11.9; HRMS (ESI) m/z 337.1668
(M + H)+ (calcd for C19H21N4O2 337.1665); HPLC (method 1)
95.9% (tR = 16.00 min). Anal. (C19H20N4O2·CF3COOH·H2O) C, H,
N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(2,6-dimethoxyphenyl)methylidene]-2,5-piperazinedione
(23). Yield of 50% from 9; mp 161−163 °C (dec); IR (KBr, cm−1)
3196, 3048, 2979, 1688, 1652, 1406; 1H NMR (300 MHz, DMSO-d6)
δ 12.52 (br s, 1H), 12.00 (br s, 1H), 9.20 (s, 1H), 8.01 (s, 1H), 7.33 (t,
J = 8.4 Hz, 1H), 6.80 (s, 1H), 6.75 (s, 1H), 6.71 (d, J = 8.4 Hz, 2H),
3.83 (s, 6H), 1.38 (s, 9H); 13C NMR (150 MHz, DMSO-d6) 158.1,
158.0, 156.8, 156.7, 140.5, 134.7, 130.8, 127.1, 110.6, 107.6, 104.6,
104.0, 56.3, 32.3, 31.0; HRMS (EI) m/z 396.1789 (M+) (calcd for
C21H24N4O4 396.1797); HPLC (method 1) 99.2% (tR = 16.22 min).
Anal. (C21H24N4O4·1.5CF3COOH·H2O) C, H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(3,4-dimethoxyphenyl)methylidene]-2,5-piperazinedione
(24). Yield of 30% from 9; mp 264−266 °C (dec); IR (KBr, cm−1)
3393, 3253, 3096, 2972, 1670, 1638, 1412; 1H NMR (300 MHz,
CDCl3) δ 12.32 (s, 1H), 9.23 (s, 1H), 8.12 (s, 1H), 7.58 (s, 1H), 7.01
(s, 2H), 6.95 (s, 2H), 6.85 (s, 1H), 3.93 (s, 3H), 3.90 (s, 3H), 1.46 (s,
9H); 13C NMR (150 MHz, DMSO-d6) 158.0, 157.0, 149.3, 149.0,
140.6, 134.8, 131.2, 126.2, 125.5, 124.4, 122.8, 115.0, 113.4, 112.3,
105.2, 56.0, 55.9, 32.4, 31.1; HRMS (EI) m/z 396.1806 (M+) (calcd
for C21H24N4O4 396.1797); HPLC (method 1) 96.4% (tR = 15.14
min). Anal. (C21H24N4O4·1.5H2O·0.5MeOH) C, H, N.
Compounds 17−65 were prepared from compound 9 according to
the procedure described for the synthesis of 11.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(2-methoxyphenyl)methylidene]-2,5-piperazinedione (17).
Yield of 29% from 9; mp 260−262 °C (dec); IR (KBr, cm−1) 3196,
2973, 1671, 1637, 1417; 1H NMR (300 MHz, CDCl3) δ 12.32 (s, 1H),
9.45 (s, 1H), 8.59 (s, 1H), 7.59 (s, 1H), 7.32−7.38 (m, 2H), 7.01 (s,
1H), 7.00 (s, 1H), 6.97−7.06 (m, 2H), 3.94 (s, 3H), 1.46 (s, 9H); 13C
NMR (150 MHz, DMSO-d6) 157.5, 157.2, 156.9, 140.6, 134.7, 130.5,
130.3, 126.8, 122.3, 121.1, 111.9, 110.7, 104.9, 56.0, 32.3, 31.0; HRMS
(EI) m/z 366.1696 (M+) (calcd for C20H22N4O3 366.1692); HPLC
(method 1) 97.2% (tR = 16.11 min). Anal. (C20H22N4O3·0.25H2O) C,
H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(3-methoxyphenyl)methylidene]-2,5-piperazinedione (18).
Yield of 41% from 9; mp 251−253 °C (dec); IR (KBr, cm−1) 3175,
2968, 1672, 1634, 1416; 1H NMR (300 MHz, CDCl3) δ 12.37 (s, 1H),
9.33 (br s, 1H), 8.11 (s, 1H), 7.58 (s, 1H), 7.26−7.39 (m, 1H), 7.02
(s, 1H), 7.00 (s, 1H), 6.97 (s, 1H), 6.86−6.92 (m, 2H), 3.84 (s, 3H),
1.46 (s, 9H); 13C NMR (100 MHz, DMSO-d6) 159.3, 157.5, 156.3,
140.4, 134.5, 134.4, 130.7, 129.8, 126.8, 123.8, 121.5, 114.5, 113.9,
113.8, 105.1, 55.1, 31.9, 30.7; HRMS (EI) m/z 366.1691 (M+) (calcd
for C20H22N4O3 366.1692); HPLC (method 3) 95.4% (tR = 18.82
min). Anal. (C20H22N4O3·CF3COOH·0.5H2O) C, H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(4-methoxyphenyl)methylidene]-2,5-piperazinedione (19).
Yield of 54% from 9; mp 268−270 °C (dec); IR (KBr, cm−1) 3409,
3248, 2975, 1668, 1622, 1608, 1406, 1344; 1H NMR (300 MHz,
CDCl3) δ 12.32 (s, 1H), 9.30 (br s, 1H), 8.06 (s, 1H), 7.58 (s, 1H)
7.35 (d, J = 8.4 Hz, 2H), 7.00 (s, 1H), 6.95 (d, J = 8.4 Hz, 2H), 6.96
(s, 1H), 3.85 (s, 3H), 1.46 (s, 9H); 13C NMR (150 MHz, DMSO-d6)
159.6, 158.0, 157.1, 140.6, 134.8, 131.4, 131.2, 126.1, 125.5, 124.4,
114.7, 114.6, 105.1, 55.7, 32.4, 31.1; HRMS (EI) m/z 366.1693 (M+)
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(3,5-dimethoxyphenyl)methylidene]-2,5-piperazinedione
(25). Yield of 22% from 9; mp 266−268 °C (dec); IR (KBr, cm−1)
1
3197, 2969, 1670, 1647, 1601, 1419; H NMR (300 MHz, CDCl3) δ
12.36 (s, 1H), 9.29 (s, 1H), 8.13 (s, 1H), 7.58 (s, 1H), 7.02 (s, 1H),
6.93 (s, 1H), 6.51 (s, 2H), 6.44 (s, 1H), 3.82 (s, 6H), 1.45 (s, 9H); 13C
NMR (100 MHz, DMSO-d6) 160.5, 157.5, 156.3, 140.4, 135.0, 134.4,
130.7, 127.0, 123.8, 114.0, 107.1, 105.2, 100.3, 55.2, 32.0, 30.7; HRMS
(EI) m/z 396.1790 (M+) (calcd for C21H24N4O4 396.1797); HPLC
( m e t h o d 1 ) 9 6 . 4 % ( t R
=
1 6 . 6 7 m i n ) . A n a l .
(C21H24N4O4·0.25CF3COOH) C, H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(2-methylphenyl)methylidene]-2,5-piperazinedione (26).
Yield of 70% from 9; mp 148−150 °C (dec); IR (KBr, cm−1) 3167,
3066, 3033, 2974, 2877, 1686, 1668, 1652, 1401; 1H NMR (300 MHz,
DMSO-d6) δ 12.57 (br s, 1H), 12.03 (s, 1H), 9.85 (s, 1H), 8.04 (s,
1H), 7.28−7.41 (m, 1H), 7.21−7.25 (m, 3H), 6.80 (s, 1H), 6.79 (s,
1H), 2.26 (s, 3H), 1.38 (s, 9H); 13C NMR (100 MHz, DMSO-d6)
157.6, 157.0, 140.5, 137.3, 134.7, 132.7, 130.7, 129.0, 128.5, 127.6,
126.5, 113.6, 104.7, 32.3, 30.9, 20.2; HRMS (EI) m/z 350.1734 (M+)
(calcd for C20H22N4O3 366.1692); HPLC (method 1) 99.1% (tR
19.20 min). Anal. (C20H22N4O3·H2O·0.5MeOH) C, H, N.
=
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(2,3-dimethoxyphenyl)methyllidene]-2,5-piperazinedione
(20). Yield of 15% from 9; mp 141−143 °C (dec); IR (KBr, cm−1)
1
3212, 2966, 1670, 1632, 1413; H NMR (300 MHz, DMSO-d6) δ
12.37 (br s, 1H), 12.22 (s, 1H), 9.73 (s, 1H), 7.88 (s, 1H) 7.02−7.17
(m, 3H), 6.85 (s, 1H), 6.76 (s, 1H), 3.84 (s, 3H), 3.71 (s, 3H) 1.35 (s,
9H); 13C NMR (100 MHz, DMSO-d6) 157.1, 156.1, 152.8, 146.2,
140.5, 134.4, 130.7, 127.3, 127.2, 124.6, 123.7, 122.1, 113.1, 109.8,
105.3, 60.7, 55.8, 32.0, 30.7; HRMS (EI) m/z 396.1790 (M+) (calcd
for C21H24N4O4 396.1797); HPLC (method 1) 95.1% (tR = 14.98
min). Anal. (C21H24N4O4·CF3COOH) C, H, N.
(calcd for C20H22N4O2 350.1743); HPLC (method 2) 95.8% (tR
15.59 min). Anal. (C20H22N4O2·0.9CF3COOH·0.5H2O) C, H, N.
=
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(3-methylphenyl)methylidene]-2,5-piperazinedione (27).
Yield of 62% from 9; mp 156−158 °C (dec); IR (KBr, cm−1) 3194,
3118, 3040, 2975, 2875, 2652, 1689, 1651, 1399; 1H NMR (300 MHz,
DMSO-d6) δ 12.66 (br s, 1H), 12.00 (br s, 1H), 10.10 (s, 1H), 8.06 (s,
1H), 7.28−7.38 (m, 3H), 7.12−7.20 (m, 1H), 6.81 (s, 1H), 6.73 (s,
1H), 2.34 (s, 3H), 1.38 (s, 9H); 13C NMR (100 MHz, DMSO-d6)
157.6, 156.4, 140.4, 137.8, 134.4, 133.1, 130.7, 129.7, 128.8, 128.6,
126.5, 126.4, 123.8, 114.1, 105.1, 32.0, 30.7, 21.1; HRMS (EI) m/z
350.1743 (M+) (calcd for C20H22N4O2 350.1743); HPLC (method 2)
97.5% (tR = 16.78 min). Anal. (C20H22N4O2·CF3COOH·0.5H2O) C,
H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(2,4-dimethoxyphenyl)methylidene]-2,5-piperazinedione
(21). Yield of 27% from 9; mp 266−268 °C (dec); IR (KBr, cm−1)
1
3397, 3259, 2969, 1663, 1637, 1417; H NMR (300 MHz, CDCl3) δ
12.26 (s, 1H), 9.41 (br s, 1H), 8.68 (s, 1H), 7.57 (s, 1H), 7.28 (s, 1H),
6.99 (s, 1H), 6.95 (s, 1H), 6.53−6.60 (m, 2H), 3.92 (s, 3H), 3.85 (s,
3H), 1.45 (s, 9H); 13C NMR (150 MHz, DMSO-d6) 161.5, 158.5,
157.5, 157.0, 140.5, 134.7, 131.5, 131.2, 125.3, 124.4, 115.0, 110.8,
106.1, 105.0, 99.1, 56.2, 55.9, 32.4, 31.1; HRMS (EI) m/z 396.1795
(M+) (calcd for C21H24N4O4 396.1797); HPLC (method 1) 97.9% (tR
= 16.74 min). Anal. (C21H24N4O4·0.75H2O·CH3OH) C, H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(4-methylphenyl)methylidene]-2,5-piperazinedione (28).
Yield of 50% from 9; mp 168−170 °C (dec); IR (KBr, cm−1) 3369,
3168, 3040, 2975, 2878, 1688, 1654, 1400; 1H NMR (300 MHz,
1065
dx.doi.org/10.1021/jm2009088 | J. Med. Chem. 2012, 55, 1056−1071

Journal of Medicinal Chemistry
Article
DMSO-d6) δ 12.60 (br s, 1H), 11.97 (s, 1H), 10.02 (s, 1H), 8.06 (s,
1H), 7.44 (d, J = 7.8 Hz, 2H), 7.24 (d, J = 7.8 Hz, 2H), 6.80 (s, 1H),
6.73 (s, 1H), 2.33 (s, 3H), 1.38 (s, 9H); 13C NMR (100 MHz, DMSO-
d6) 157.5, 156.9, 140.3, 138.0, 134.5, 130.6, 130.5, 129.5, 129.5, 128.3,
126.0, 114.7, 32.1, 30.6, 21.2; HRMS (EI) m/z 350.1734 (M+) (calcd
for C20H22N4O2 350.1743); HPLC (method 1) >99.9% (tR = 16.91
min). Anal. (C20H22N4O2·1.2CF3COOH) C, H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(2,6-dimethylphenyl)methylidene]-2,5-piperazinedione (29).
Yield of 51% from 9; mp 158−160 °C (dec); IR (KBr, cm−1) 3434,
3174, 3063, 3034, 2973, 2876, 1692, 1670, 1649, 1405; 1H NMR (300
MHz, DMSO-d6) δ 12.63 (br s, 1H), 11.98 (br s, 1H), 9.67 (s, 1H),
8.10 (s, 1H), 7.04−7.29 (m, 3H), 6.78 (s, 1H), 6.73 (s, 1H), 2,16 (s,
6H), 1.37 (s, 9H); 13C NMR (100 MHz, DMSO-d6) 157.5, 156.6,
140.5, 136.6, 134.7, 132.1, 128.6, 128.0, 127.8, 114.4, 32.3, 30.8, 20.5;
HRMS (EI) m/z 364.1894 (M+) (calcd for C21H24N4O2 364.1899);
7.57 (m, 2H), 7.30−7.38 (m, 1H), 7.15−7.19 (m, 1H), 7.04 (s, 1H),
6.96 (s, 1H), 1.43 (s, 9H); 13C NMR (150 MHz, DMSO-d6) 157.8,
156.6, 140.8, 134.8, 133.5, 132.2, 131.1, 130.0, 129.9, 128.9, 127.8,
110.6, 105.4, 32.4, 31.0; HRMS (EI) m/z 370.1191 (M+) (calcd for
C19H19N4O2Cl 370.1196); HPLC (method 1) >99.9% (tR = 16.56
min). Anal. (C19H19N4O2Cl·CF3COOH·H2O) C, H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(3-chlorophenyl)methylidene]-2,5-piperazinedione (35).
Yield of 76% from 9; mp 276−278 °C (dec); IR (KBr, cm−1) 3185,
3114, 3072, 3039, 2978, 2876, 2656, 1690, 1655, 1402; 1H NMR (270
MHz, CDCl3) δ 12.53 (s, 1H), 11.61 (br s, 1H), 8.48 (s, 1H), 7.55 (s,
1H), 7.18−7.43 (m, 4H), 7.06 (s, 1H), 6.86 (s, 1H), 1.44 (s, 9H); 13C
NMR (100 MHz, DMSO-d6) 157.7, 156.6, 140.4, 135.6, 134.5, 133.4,
130.5, 129.0, 128.3, 127.9, 127.8, 112.8, 104.6, 32.1, 30.6; HRMS (EI)
m/z 370.1188 (M+) (calcd for C19H19N4O2Cl 370.1196); HPLC
( m e t h o d 2 ) 9 8 . 2 % ( t R
=
1 7 . 4 6 m i n ) . A n a l .
HPLC (method 1) >99.9% (tR
(C21H24N4O2·0.25CF3COOH) C, H, N.
=
16.71 min). Anal.
(C19H19N4O2Cl·CF3COOH·H2O) C, H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(4-chlorophenyl)methylidene]-2,5-piperazinedione (36).
Yield of 55% from 9; mp 166−168 °C (dec); IR (KBr, cm−1) 3166,
3041, 2974, 2876, 2673, 1688, 1656, 1400; 1H NMR (270 MHz,
CDCl3) δ 12.51 (br s, 1H), 11.62 (br s, 1H), 8.41 (s, 1H), 7.55 (s,
1H), 7.33−7.47 (m, 4H), 7.05 (s, 1H), 6.87 (s, 1H), 1.44 (s, 9H); 13C
NMR (100 MHz, DMSO-d6) 157.7, 156.2, 140.5, 134.4, 132.4, 132.3,
131.1, 130.7, 128.6, 127.3, 123.8, 112.6, 105.3, 32.0, 30.7; HRMS (EI)
m/z 370.1201 (M+) (calcd for C19H19N4O2Cl 370.1196); HPLC
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(2-fluorophenyl)methylidene]-2,5-piperazinedione (30).
Yield of 68% from 9; mp 142−145 °C (dec); IR (KBr, cm−1) 3174,
3047, 2676, 2877, 1691, 1653, 1399; 1H NMR (300 MHz, DMSO-d6)
δ 12.58 (br s, 1H), 12.09 (br s, 1H), 10.28 (s, 1H), 8.04 (s, 1H), 7.57
(t, J = 7.8 Hz, 1H), 7.34−7.43 (m, 1H), 7.21−7.29 (m, 2H), 6.83 (s,
1H), 6.70 (s, 1H), 1.38 (s, 9H); 13C NMR (100 MHz, DMSO-d6)
161.5, 159.0, 157.7, 156.6, 140.7, 134.8, 131.1, 130.6, 130.5, 128.8,
125.0, 125.0, 121.6, 121.4, 116.3, 116.1, 107.0, 105.2, 32.3, 30.9;
HRMS (EI) m/z 354.1501 (M+) (calcd for C19H19N4O2F 354.1492);
( m e t h o d 1 ) 9 5 . 8 % ( t R
=
1 7 . 2 4 m i n ) . A n a l .
(C19H19ClN4O2·0.5CF3COOH) C, H, N.
HPLC (method 1) 99.8% (tR
(C19H19N4O2F·CF3COOH·0.5H2O) C, H, N.
=
15.52 min). Anal.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(2,3-dichlorophenyl)methylidene]-2,5-piperazinedione (37).
Yield of 27% from 9; mp 165−168 °C (dec); IR (KBr, cm−1) 3181,
3048, 2975, 2874, 1692, 1656, 1404; 1H NMR (300 MHz, DMSO-d6)
δ 12.54 (br s, 1H), 12.17 (br s, 1H), 10.40 (s, 1H), 8.01 (s, 1H), 7.37−
7.60 (m, 3H), 6.84 (s, 1H), 6.72 (s, 1H), 1.38 (s, 9H); 13C NMR (150
MHz, DMSO-d6) 157.9, 156.3, 140.9, 135.0, 134.9, 132.4, 131.3, 130.1,
129.9, 129.7, 128.8, 110.4, 110.0, 105.8, 32.4, 31.0; HRMS (EI) m/z
404.0801 (M+) (calcd for C19H18N4O2Cl2 404.0806); HPLC (method
1) 98.8% (tR = 18.52 min). Anal. (C19H18Cl2N4O2·CF3COOH·H2O)
C, H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(2-bromophenyl)methylidene]-2,5-piperazinedione (38).
Yield of 52% from 9; mp 146−148 °C (dec); IR (KBr, cm−1) 3182,
3041, 2973, 2873, 1692, 1656, 1399; 1H NMR (300 MHz, DMSO-d6)
δ 12.58 (br s, 1H), 12.10 (br s, 1H), 10.33 (s, 1H), 8.04 (s, 1H), 7.69
(d, J = 7.5 Hz, 1H), 7.57 (d, J = 7.2 Hz, 1H), 7.44 (t, J = 7.2 Hz, 1H),
7.27 (t, J = 7.5 Hz, 1H), 6.82 (s, 1H), 6.72 (s, 1H), 1.38 (s, 9H); 13C
NMR (100 MHz, DMSO-d6) 157.5, 156.4, 140.5, 134.5, 133.7, 132.8,
131.0, 130.0, 128.4, 128.1, 124.1, 112.9, 32.1, 104.8, 32.1, 30.6; HRMS
(EI) m/z 414.0694 (M+) (calcd for C19H19N4O2Br 414.0691); HPLC
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(3-fluorophenyl)methylidene]-2,5-piperazinedione (31).
Yield of 63% from 9; mp 169−171 °C (dec); IR (KBr, cm−1) 3419,
3184, 3124, 3048, 1692, 1661, 1624; 1H NMR (300 MHz, DMSO-d6)
δ 12.59 (br s, 1H), 12.05 (br s, 1H), 10.32 (s, 1H), 8.05 (s, 1H), 7.30−
7.49 (m, 3H), 7.10−7.19 (m, 1H), 6.82 (s, 1H), 6.74 (s, 1H), 1.38 (s,
9H); 13C NMR (100 MHz, DMSO-d6) 163.4, 161.0, 157.7, 156.2,
140.5, 135.7, 135.6, 134.4, 130.7, 130.6, 130.5, 127.6, 125.6, 125.5,
123.7, 115.9, 115.7, 114.8, 114.6, 112.4, 105.3, 32.0, 30.7; HRMS (EI)
m/z 354.1493 (M+) (calcd for C19H19N4O2F 354.1492); HPLC
( m e t h o d 3 ) 9 9 . 3 % ( t R
=
1 8 . 5 9 m i n ) . A n a l .
(C19H19N4O2F·CF3COOH·H2O) C, H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(4-fluorophenyl)methylidene]-2,5-piperazinedione (32).
Yield of 83% from 9; mp 168−170 °C (dec); IR (KBr, cm−1) 3179,
3045, 2973, 2878, 2669, 1689, 1655, 1623, 1402; 1H NMR (300 MHz,
DMSO-d6) δ 12.60 (br s, 1H), 12.00 (br s, 1H), 10.22 (s, 1H), 8.06 (s,
1H), 7.52−7.61 (dd, J = 5.7, 8.4 Hz, 2H), 7.25 (t, J = 8.7 Hz, 2H), 6.81
(s, 1H), 6.75 (s, 1H), 1.38 (s, 9H); 13C NMR (100 MHz, DMSO-d6)
163.3, 160.8, 157.9, 157.1, 140.5, 134.7, 132.0, 132.0, 130.1, 130.1,
126.9, 116.2, 116.0, 113.8, 104.6, 32.3, 30.9; HRMS (EI) m/z
354.1488 (M+) (calcd for C19H19N4O2F 354.1492); HPLC (method
( m e t h o d 1 ) 9 5 . 4 % ( t R
=
1 6 . 8 0 m i n ) . A n a l .
(C19H19BrN4O2·CF3COOH·0.5H2O) C, H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(3-bromophenyl)methylidene]-2,5-piperazinedione (39).
Yield of 55% from 9; mp 173−175 °C (dec); IR (KBr, cm−1) 3186,
3113, 3038, 2978, 2876, 1689, 1656, 1404; 1H NMR (300 MHz,
DMSO-d6) δ 12.58 (br s, 1H), 12.05 (br s, 1H), 10.46 (s, 1H), 8.04 (s,
1H), 7.70 (s, 1H), 7.44−7.55 (m, 2H), 7.35 (t, J = 7.8 Hz, 1H), 6.82
(s, 1H), 6.71 (s, 1H), 1.38 (s, 9H); 13C NMR (150 MHz, DMSO-d6)
158.1, 156.7, 140.8, 136.2, 134.8, 132.0, 131.0, 128.9, 128.1, 122.4,
112.7, 105.4, 32.4, 31.0; HRMS (EI) m/z 414.0687 (M+) (calcd for
C19H19N4O2Br 414.0691); HPLC (method 1) 99.4% (tR = 17.79 min).
Anal. (C19H19N4O2Br·CF3COOH·0.25H2O) C, H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(4-bromophenyl)methylidene]-2,5-piperazinedione (40).
Yield of 50% from 9; mp 176−178 °C (dec); IR (KBr, cm−1) 3383,
3206, 3039, 2971, 2873, 2660, 1687, 1656, 1625, 1397; 1H NMR (300
MHz, DMSO-d6) δ 12.60 (br s, 1H), 12.03 (br s, 1H), 10.28 (s, 1H),
8.06 (s, 1H), 7.60 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.1 Hz, 2H), 6.81
(s, 1H), 6.70 (s, 1H), 1.38 (s, 9H); 13C NMR (150 MHz, DMSO-d6)
157.9, 156.9, 140.6, 134.7, 133.0, 132.0, 131.8, 127.6, 121.5, 113.4,
1 )
9 5 . 4 %
( t
=
1 5 . 5 9
m i n ) .
A n a l .
R
(C19H19N4O2F·CF3COOH·0.75H2O) C, H, N.
(3Z,6Z)-3-((5-tert-Butyl-1H-imidazol-4-yl)methylene)-6-(2,5-
difluorobenzylidene)piperazine-2,5-dione (33). Yield of 75%
from 9; mp >290 °C; IR (KBr, cm−1) 3406, 3192, 3117, 2979, 2875,
1
1693, 1655, 1398; H NMR (300 MHz, CDCl3) δ 12.62 (br s, 1H),
12.09 (br s, 1H), 10.51 (s, 1H), 8.07 (s, 1H), 7.21−7.41 (m, 3H), 6.84
(s, 1H), 6.63 (s, 1H), 1.38 (s, 9H); 13C NMR (100 MHz, DMSO-d6)
159.0, 158.3, 158.0, 157.3, 157.2, 156.6, 155.8, 154.7, 140.2, 134.2,
129.2, 122.5, 122.4, 122.3, 122.2, 117.0, 116.7, 116.3, 116.2, 116.1,
116.0, 105.1, 104.8, 31.8, 30.3; HRMS (EI) m/z 372.1934 (M+) (calcd
for C19H18N4O2F2 372.1397); HPLC (method 1) >99.9% (tR = 20.14
min). Anal. (C19H18F2N4O2·CF3COOH) C, H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(2-chlorophenyl)methylidene]-2,5-piperazinedione (34).
Yield of 42% from 9; mp 160−162 °C (dec); IR (KBr, cm−1) 3166,
1
3041, 2973, 2874, 1689, 1668, 1399; H NMR (270 MHz, CDCl3) δ
12.52 (br s, 1H), 11.60 (br s, 1H), 10.48 (s, 1H), 8.35 (s, 1H), 7.43−
1066
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Journal of Medicinal Chemistry
Article
104.8, 32.3, 30.9; HRMS (EI) m/z 414.0698 (M+) (calcd for
C19H19N4O2Br 414.0691); HPLC (method 1) 99.1% (tR = 17.87 min).
Anal. (C19H19BrN4O2·CF3COOH·0.5H2O) C, H, N.
105.1, 63.0, 40.1, 39.9, 39.7, 39.5, 39.3, 39.1, 38.9, 31.9, 30.7, 14.7;
HRMS (EI) m/z 380.1843 (M+) (calcd for C21H24N4O3 380.1848);
HPLC (method 1) 98.8% (tR
= 17.46 min). Anal.
(C21H24N4O3·0.25H2O·CF3COOH) C, H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(2-nitrophenyl)methylidene]-2,5-piperazinedione (41). Yield
of 22% from 9; mp 168−170 °C (dec); IR (KBr, cm−1) 3384, 3189,
3053, 2973, 2872, 2664, 1691, 1657, 1407; 1H NMR (300 MHz,
DMSO-d6) δ 12.34 (br s, 1H), 12.31 (s, 1H), 10.35 (s, 1H), 8.14 (d, J
= 8.1 Hz, 1H), 7.87 (s, 1H), 7.78 (t, J = 7.5 Hz, 1H), 7.64 (d, J = 7.8
Hz, 1H), 7.59 (t, J = 8.1 Hz, 1H), 6.93 (s, 1H), 6.86 (s, 1H), 1.38 (s,
9H); 13C NMR (150 MHz, DMSO-d6) 157.8, 156.5, 148.2, 140.7,
134.8, 134.6, 132.1, 129.7, 129.5, 129.0, 125.3, 111.2, 105.2, 32.3, 30.9;
HRMS (EI) m/z 381.1429 (M+) (calcd for C19H19N5O4 381.1437);
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(3-cyanophenyl)methylidene]-2,5-piperazinedione (47).
Yield of 41% from 9; mp 163−165 °C (dec); IR (KBr, cm−1) 3190,
3114, 3073, 3040, 2978, 2876. 1691, 1656, 1629, 1409; 1H NMR (300
MHz, DMSO-d6) δ 12.58 (br s, 1H), 12.10 (br s, 1H), 10.58 (s, 1H),
8.04 (s, 1H), 7.98 (s, 1H), 7.76 (t, J = 8.6 Hz, 2H), 7.58 (t, J = 7.8 Hz,
1H), 6.84 (s, 1H), 6.74 (s, 1H), 1.38 (s, 9H); 13C NMR (150 MHz,
DMSO-d6) 158.2, 156.6, 140.9, 134.9, 134.8, 134.6, 133.2, 131.6,
130.1, 128.7, 119.3, 112.2, 111.9, 105.4, 32.4, 31.0; HRMS (EI) m/z
361.1536 (M+) (calcd for C20H19N5O2 361.1538); HPLC (method 1)
HPLC (method 1) 95.1% (tR
= 14.40 min). Anal.
9 5 . 0 %
( t
=
1 4 . 8 3
m i n ) .
A n a l .
(C19H19N5O4·CF3COOH·0.25H2O) C, H, N.
R
(C20H19N5O2·CF3COOH·0.5CH3COOCH2CH3) C, H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(3-nitrophenyl)methylidene]-2,5-piperazinedione (42). Yield
of 50% from 9; mp 273−275 °C (dec); IR (KBr, cm−1) 3214, 3086,
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(3-vinylphenyl)methylidene]-2,5-piperazinedione (48). Yield
of 67% from 9; mp 183−185 °C (dec); IR (KBr, cm−1) 3434, 3172,
3036, 2971, 2873, 1689, 1655, 1619, 1402; 1H NMR (300 MHz,
DMSO-d6) δ 12.64 (br s, 1H), 11.98 (br s, 1H), 10.24 (s, 1H), 8.09 (s,
1H), 7.59 (s, 1H), 7.36−7.48 (m, 3H), 6.81 (s, 1H), 6.77 (s, 1H), 6.77
(dd, J = 11, 18 Hz, 1H), 5.89 (d, J = 18 Hz, 1H), 5.31 (d, J = 11 Hz,
1H), 1.38 (s, 9H); 13C NMR (150 MHz, DMSO-d6) 157.9, 157.0,
140.6, 137.9, 136.9, 134.7, 134.0, 129.4, 129.2, 127.7, 127.2, 126.1,
115.3, 114.5, 104.9, 32.3, 30.9; HRMS (EI) m/z 362.1740 (M+) (calcd
for C21H22N4O2 362.1743); HPLC (method 1) 96.7% (tR = 17.70
min). Anal. (C21H22N4O2·CF3COOH·0.5H2O) C, H, N.
1
2967, 1675, 1633, 1414, 1348; H NMR (300 MHz, DMSO-d6) δ
12.50 (br s, 1H), 12.17 (br s, 1H), 10.64 (s, 1H), 8.29 (s, 1H), 7.82−
8.16 (m, 3H), 7.67 (t, J = 7.8 Hz, 1H), 6.86 (s, 1H), 6.82 (s, 1H), 1.39
(s, 9H); 13C NMR (100 MHz, DMSO-d6) 179.9, 157.9, 155.9, 147.9,
140.6, 135.9, 135.0, 134.5, 130.7, 129.9, 128.8, 123.9, 123.7, 122.3,
111.1, 105.5, 32.0, 30.7; HRMS (EI) m/z 381.1430 (M+) (calcd for
C19H19N5O4 381.1437); HPLC (method 1) 99.2% (tR = 15.70 min).
Anal. (C19H19N5O4·CF3COOH) C, H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(4-nitrophenyl)methylidene]-2,5-piperazinedione (43). Yield
of 18% from 9; mp 153−155 °C (dec); IR (KBr, cm−1) 3411, 3300,
2957, 1672, 1630, 1417, 1336, 1108; 1H NMR (300 MHz, DMSO-d6)
δ 12.43 (br s, 1H), 12.29 (s, 1H), 10.54 (s, 1H), 8.23 (d, J = 9 Hz,
2H), 7.92 (s, 1H), 7.76 (d, J = 8.7 Hz, 2H), 6.88 (s, 1H), 6.80 (s, 1H),
1.39 (s, 9H); 13C NMR (150 MHz, DMSO-d6) 158.2, 156.2, 146.3,
141.2, 141.0, 134.8, 131.1, 130.7, 124.1, 124.0, 111.2, 106.0, 32.3, 31.0;
HRMS (EI) m/z 381.1442 (M+) (calcd for C19H19N5O4 381.1437);
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-{(Z)-
1-[3-(trifluoromethyl)phenyl]methylidene}-2,5-piperazine-
dione (49). Yield of 56% from 9; mp 271−273 °C (dec); IR (KBr,
cm−1) 3180, 3115, 3073, 3038, 2977, 2879, 1690. 1652, 1407; 1H
NMR (300 MHz, DMSO-d6) δ 12.59 (br s, 1H), 12.08 (br s, 1H),
10.57 (s, 1H), 8.05 (s, 1H), 7.75−7.85 (m, 2H), 7.59−7.68 (m, 2H),
6.83 (s, 1H), 6.80 (s, 1H) 1.38 (s, 9H); 13C NMR (150 MHz, DMSO-
d6) 158.1, 156.8, 140.7, 134.8, 133.7, 130.0, 129.8, 129.6, 128.4, 126.2,
126.2, 125.5, 124.8, 124.8, 123.7, 112.8, 105.1, 32.3, 30.9; HRMS (EI)
m/z 404.1466 (M+) (calcd for C20H19N4O2F3 404.1460); HPLC
HPLC (method 2) 96.4% (tR
= 15.32 min). Anal.
(C19H19N5O4·0.4CF3COOH·CH3COOCH2CH3) C, H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(2-hydroxyphenyl)methylidene]-2,5-piperazinedione (44).
Yield of 67% from 9; mp 220−222 °C (dec); IR (KBr, cm−1) 3134,
2981, 2613, 1646, 1434, 1401, 1362; 1H NMR (300 MHz, DMSO-d6)
δ 12.61 (br s, 1H), 11.95 (br s, 1H), 10.85 (s, 1H), 10.16 (s, 1H), 8.07
(s, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.21 (t, J = 7.8 Hz, 1H), 6.94 (d, J =
7.8 Hz, 1H), 6.88 (t, J = 7.8 Hz, 1H), 6.79 (s, 1H), 6.75 (s, 1H), 1.37
(s, 9H); 13C NMR (150 MHz, DMSO-d6) 157.1, 156.9, 154.5, 140.4,
134.7, 132.5, 130.6, 125.8, 121.0, 120.4, 116.7, 112.7, 104.3, 32.3, 30.8;
HRMS (EI) m/z 352.1539 (M+) (calcd for C19H20N4O3 352.1535);
( m e t h o d 1 ) 9 9 . 6 % ( t R
(C20H19F3N4O2·CF3COOH) C, H, N.
=
1 8 . 3 2 m i n ) . A n a l .
(3Z,6Z)-3-(3-Benzoylbenzylidene)-6-((5-tert-butyl-1H-imida-
zol-4-yl)methylene)piperazine-2,5-dione (50). 3-Benzoylbenzal-
dehyde was synthesized as previously reported.31 Yield of 26% from 9;
mp 158−161 °C; IR (KBr, cm−1) 3161, 3065, 2972, 2874, 1685, 1654,
1397; 1H NMR (300 MHz, DMSO-d6) δ 12.47 (br s, 1H), 12.15 (br s,
1H), 10.38 (s, 1H), 7.96 (br s, 1H), 7.54−7.83 (m, 9H), 6.80 (s, 1H),
6.83 (s, 1H), 1.38 (s, 9H); 13C NMR (150 MHz, DMSO-d6) 196.0,
158.0, 156.7, 140.8, 137.8, 137.3, 134.8, 134.0, 133.8, 133.3, 130.6,
130.3, 129.4, 129.3, 129.1, 128.1, 113.4, 105.3, 32.4, 31.0; HRMS (EI)
m/z 440.1845 (M+) (calcd for C26H24N4O3 440.1848); HPLC
(method 1) 96.1% (tR = 18.67 min). Anal. (C26H24N4O3·CF3COOH)
C, H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(2-chloro-6-fluorophenyl)methylidene]-2,5-piperazinedione
(51). Yield of 40% from 9; mp 225−227 °C (dec); IR (KBr, cm−1)
3189, 3049, 2974, 2875, 1693, 1654, 1406; 1H NMR (300 MHz,
DMSO-d6) δ 12.62 (br s, 1H), 12.13 (br s, 1H), 10.51 (s, 1H), 8.07 (s,
1H), 7.35−7.46 (m, 2H), 7.23−7.31 (m, 1H), 6.84 (s, 1H), 6.53 (s,
1H), 1.38 (s, 9H); 13C NMR (150 MHz, DMSO-d6) 161.4, 159.8,
157.6, 156.0, 140.9, 134.8, 134.5, 134.5, 130.9, 130.8, 130.8, 125.7,
121.1, 121.0, 115.5, 115.3, 105.8, 104.6, 32.4, 30.9; HRMS (EI) m/z
388.1096 (M+) (calcd for C19H18N4O2ClF 388.1102); HPLC (method
HPLC (method 1) >99.9% (tR
(C19H20N4O3·CF3COOH) C, H, N.
= 14.46 min). Anal.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(3-hydroxyphenyl)methylidene]-2,5-piperazinedione (45).
Yield of 32% from 9; mp 171−173 °C (dec); IR (KBr, cm−1) 3167,
3094, 2965, 2879, 1683, 1611, 1423, 1403; 1H NMR (300 MHz,
DMSO-d6) δ 12.59 (br s, 1H), 12.01 (br s, 1H), 9.92 (s, 1H), 8.04 (s,
1H), 7.22 (t, J = 7.8 Hz, 1H), 6.93 (d, J = 7.8 Hz, 1H), 6.90 (s, 1H),
6.81 (s, 1H), 6.74 (d, J = 7.8 Hz, 1H), 6.67 (s, 1H), 1.38 (s, 9H); 13C
NMR (100 MHz, DMSO-d6) 157.5, 157.4, 156.3, 140.4, 134.4, 130.7,
129.8, 126.5, 123.8, 120.1, 115.6, 115.4, 114.1, 105.1, 32.0, 30.7;
HRMS (EI) m/z 352.1534 (M+) (calcd for C19H20N4O3 352.1535);
HPLC (method 3) 97.9% (tR
= 13.54 min). Anal.
(C19H20N4O3·CF3COOH·0.5H2O) C, H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(3-ethoxyphenyl)methylidene]-2,5-piperazinedione (46).
Yield of 28% from 9; mp 158−160 °C (dec); IR (KBr, cm−1) 2975,
2874, 1687, 1649, 1396; 1H NMR (300 MHz, DMSO-d6) δ 12.58 (br
s, 1H), 12.01 (br s, 1H), 10.10 (s, 1H), 8.04 (s, 1H), 7.32 (t, J = 7.8
Hz, 1H), 7.03−7.10 (m, 2H), 6.89 (d, J = 8.4 Hz, 1H), 6.81 (s, 1H),
6.73 (s, 1H) 4.06 (q, J = 6.9 Hz, 2H), 1.38 (s, 9H), 1.35 (t, J = 6.9 Hz,
3H); 13C NMR (100 MHz, DMSO-d6) 158.6, 157.5, 156.3, 140.4,
134.5, 134.4, 130.7, 129.8, 126.8, 123.8, 121.5, 115.0, 114.3, 113.9,
1 )
9 6 . 4 %
( t
=
2 0 . 7 1
m i n ) .
A n a l .
R
(C19H18ClFN4O2·CF3COOH·0.25H2O) C, H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(1-naphthyl)methylidene]-2,5-piperazinedione (52). Yield of
36% from 9; mp 188−190 °C (dec); IR (KBr, cm−1) 3434, 3178, 3044,
1
2972, 2873, 1686, 1650, 1401; H NMR (300 MHz, DMSO-d6) δ
12.60 (br s, 1H), 12.11 (br s, 1H), 10.08 (s, 1H), 8.07 (s, 1H), 7.88−
8.01 (m, 3H), 7.53−7.65 (m, 4H), 7.21 (s, 1H), 6.81 (s, 1H), 1.38 (s,
9H); 13C NMR (150 MHz, DMSO-d6) 157.8, 156.7, 140.7, 134.8,
1067
dx.doi.org/10.1021/jm2009088 | J. Med. Chem. 2012, 55, 1056−1071

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Article
133.8, 131.7, 130.6, 129.0, 129.0, 128.8, 127.6, 127.0, 126.6, 126.3,
141.6, 141.4, 135.1, 133.4, 127.7, 126.5, 124.2, 108.1, 106.8, 32.5, 31.0;
HRMS (EI) m/z 337.1531(M+) (calcd for C18H19N2O2 337.1538).;
124.8, 112.0, 110.0, 105.3, 32.4, 31.0; HRMS (EI) m/z 386.1751 (M+)
HPLC (method 2) 98.3% (tR
= 17.64 min). Anal.
(calcd for C23H22N4O2 386.1743); HPLC (method 1) 95.4% (tR
=
(C18H19N5O2·2CF3COOH·H2O) C, H, N.
18.12 min). Anal. (C23H22N4O2·CF3COOH) C, H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(2-furyl)methylidene]-2,5-piperazinedione (59). Yield of 45%
from 9; mp 245−247 °C; IR (KBr, cm−1) 3123, 3034, 2973, 2875,
1686, 1648, 1416, 1394; 1H NMR (300 MHz, DMSO-d6) δ 12.48 (br
s, 1H), 12.20 (br s, 1H), 9.38 (s, 1H), 7.93 (s, 2H), 6.88 (s, 1H), 6.86
(s, 1H), 6.70−6.65 (m, 1H), 6.63 (s, 1H), 1.38 (s, 9H); 13C NMR
(150 MHz, DMSO-d6) 157.1, 156.3, 150.5, 145.3, 140.9, 134.8, 124.5,
114.7, 112.9, 105.3, 101.5, 32.4, 31.0; HRMS (EI) m/z 326.1373 (M+)
(calcd for C17H18N4O3 326.1379); HPLC (method 1) >99.9% (tR =
14.72 min). Anal. (C17H18N4O3·CF3COOH·0.75H2O) C, H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(5-methyl-2-furyl)methylidene]-2,5-piperazinedione (60).
Yield of 19% from 9; mp 271−273 °C (dec); IR (KBr, cm−1) 3166,
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(2-naphthyl)methylidene]-2,5-piperazinedione (53). Yield of
53% from 9; mp 158−160 °C (dec); IR (KBr, cm−1) 3394, 3178, 3042,
1
2974, 2873, 1686, 1651, 1401; H NMR (300 MHz, DMSO-d6) δ
12.64 (br s, 1H), 12.01 (br s, 1H), 10.39 (s, 1H), 8.11 (s, 1H), 8.04−
8.15 (m, 1H), 7.98−7.98 (m, 3H). 7.50−7.64 (m, 3H), 6.92 (s, 1H),
6.83 (s, 1H), 1.39 (s, 9H); 13C NMR (100 MHz, DMSO-d6) 157.7,
156.4, 140.4, 134.4, 133.0, 132.4, 130.9, 130.8, 128.5, 128.3, 128.0,
127.5, 127.2, 127.0, 126.6, 126.4, 123.9, 114.0, 105.1, 32.0, 30.7;
HRMS (EI) m/z 386.1737 (M+) (calcd for C23H22N4O2 386.1743);
HPLC (method 1) 99.2% (tR
(C23H22N4O2·CF3COOH·H2O) C, H, N.
= 18.98 min). Anal.
(3Z,6Z)-3-((5-tert-Butyl-1H-imidazol-4-yl)methylene)-6-(qui-
nolin-8-ylmethylene)piperazine-2,5-dione (54). Yield of 32%
from 9; mp >250 °C; IR (KBr, cm−1) 3435, 3241, 2962, 1656, 1633,
1415; 1H NMR (300 MHz, DMSO-d6) δ 12.31 (s, 2H), 8.99 (dd, J =
1.0, 4.6 Hz, 1H), 8.57 (dd, J = 1.0, 7.8 Hz, 1H), 8.04 (d, J = 8.0 Hz,
1H), 8.00 (d, J = 6.8 Hz, 1H), 7.88 (s, 1H), 7.75−7.66 (m, 2H), 7.15
(s, 1H), 6.88 (s, 1H), 1.39 (s, 9H); 13C NMR (150 MHz, DMSO-d6)
156.7, 156.4, 148.4, 143.9, 140.2, 138.4, 134.9, 134.3, 132.8, 130.7,
129.2, 129.0, 127.2, 126.9, 124.1, 122.0, 112.0, 109.4, 104.7, 31.8, 30.5;
HRMS (EI) m/z 387.1697 (M+) (calcd for C22H21N5O2 387.1695);
1
2976, 2874, 1691, 1663, 1653, 1404; H NMR (300 MHz, CDCl3) δ
12.44 (s, 1H), 11.70 (br s, 1H), 9.08 (s, 1H), 7.55 (s, 1H), 7.03 (s,
1H), 6.62 (s, 1H), 6.46 (d, J = 3.0 Hz, 1H), 6.14 (d, J = 2.2 Hz, 1H),
2.45 (s, 3H), 1.45 (s, 9H); 13C NMR (150 MHz, DMSO-d6) 157.1,
156.4, 154.9, 149.2, 140.8, 134.8, 123.3, 116.2, 109.4, 105.3, 101.8,
32.4, 31.0, 14.2; HRMS (EI) m/z 340.1539 (M+) (calcd for
C18H20N4O3 340.1535); HPLC (method 1) 99.1% (tR = 15.96 min).
Anal. (C18H20N4O3·CF3COOH) C, H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(5-chloro-2-furyl)methylidene]-2,5-piperazinedione (61).
Yield of 24% from 9; mp 296−298 °C (dec); IR (KBr, cm−1) 3165,
3032, 2974, 2874, 2658, 1693, 1661, 1627, 1404; 1H NMR (300 MHz,
CDCl3) δ 12.49 (s, 1H), 11.90 (br s, 1H), 8.89 (s, 1H), 7.58 (s, 1H),
7.04 (s, 1H), 6.59 (s, 1H), 6.60 (d, J = 3.2 Hz, 1H), 6.37 (d, J = 3.2
Hz, 1H), 1.45 (s, 9H); 13C NMR (150 MHz, DMSO-d6) 157.3, 156.2,
150.4, 141.0, 136.7, 134.9, 125.0, 116.3, 110.2, 105.7, 100.7, 32.4, 31.0;
HRMS (EI) m/z 360.0991 (M+) (calcd for C17H17ClN4O3 360.0989);
HPLC (method 1) >99.9% (tR
(C22H21N5O2·2H2O) C, H, N.
= 16.00 min). Anal.
(3Z,6Z)-3-((5-tert-Butyl-1H-imidazol-4-yl)methylene)-6-(iso-
quinolin-5-ylmethylene)piperazine-2,5-dione (55). Yield of 11%
from 9; mp 204−206 °C; IR (KBr, cm−1) 3179, 3042, 2975, 2875,
1692, 1671, 1415, 1405; 1H NMR (300 MHz, DMSO-d6) δ 12.48 (br
s, 1H), 12.31 (br s, 1H), 10.31 (s, 1H), 9.69 (s, 1H), 8.62 (d, J = 6.4
Hz, 1H), 8.34 (d, J = 8.1 Hz, 1H), 8.14 (d, J = 6.2 Hz, 1H), 8.07 (d, J
= 7.1 Hz, 1H), 7.96 (s, 1H), 7.92 (t, J = 7.8 Hz, 1H), 7.12 (s, 1H), 6.85
(s, 1H), 1.38 (s, 9H); 13C NMR (150 MHz, DMSO-d6) 158.0, 156.3,
150.5, 140.9, 137.9, 135.8, 134.9, 131.0, 130.6, 129.5, 129.5, 128.6,
124.7, 120.7, 109.3, 105.6, 32.4, 31.0; HRMS (EI) m/z 387.1696 (M+)
(calcd for C22H21N5O2 378.1695); HPLC (method 1) >99.9% (tR =
12.36 min). Anal. (C22H21N5O2·2CF3COOH·H2O) C, H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(2-pyridyl)methylidene]-2,5-piperazinedione (56). Yield of
51% from 9; mp 270−272 °C (dec); IR (KBr, cm−1) 3496, 3448,
3148, 3083, 2965, 1680, 1644, 1416; 1H NMR (300 MHz, DMSO-d6)
δ 12.66 (br s, 1H), 12.51 (s, 1H), 12.18 (s, 1H), 8.74 (d, J = 3.4 Hz,
1H), 8.06 (s, 1H), 7.91−8.00 (m, 1H), 7.66 (d, J = 8.1 Hz, 1H), 7.38−
7.45 (m, 1H), 6.88 (s, 1H), 6.70 (s, 1H), 1.39 (s, 9H); 13C NMR (150
MHz, DMSO-d6) 157.2, 155.9, 155.3, 149.0, 141.4, 138.2, 135.0, 131.8,
126.9, 122.8, 107.4, 106.3, 32.5, 31.0; HRMS (EI) m/z 337.1533 (M+)
HPLC (method 1) 96.8% (tR
= 16.35 min). Anal.
(C17H17ClN4O3·CF3COOH) C, H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(3-furyl)methylidene]-2,5-piperazinedione (62). Yield of 43%
from 9; mp 250−252 °C (dec); IR (KBr, cm−1) 3132, 3048, 2974,
1678, 1644, 1390; 1H NMR (270 MHz, CDCl3) δ 12.44 (s, 1H), 11.54
(br s, 1H), 8.13 (s, 1H), 7.77 (d, J = 0.8 Hz, 1H), 7.53 (br s, 2H), 7.05
(s, 1H), 6.75 (s, 1H), 6.63 (d, J = 1.1 Hz, 1H), 1.44 (s, 9H); 13C NMR
(150 MHz, DMSO-d6) 157.8, 157.1, 144.5, 144.4, 140.5, 134.7, 125.7,
118.9, 111.1, 106.3, 104.6, 32.3, 30.8; HRMS (EI) m/z 326.1385 (M+)
(calcd for C17H18N4O3 326.1379); HPLC (method 2) 95.0% (tR
10.44 min). Anal. (C17H18N4O3·CF3COOH) C, H, N.
=
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(2-thienyl)methylidene]-2,5-piperazinedione (63). Yield of
42% from 9; mp 274−276 °C; IR (KBr, cm−1) 3177, 3114, 2973,
1
2875, 1686, 1673, 1653, 1396; H NMR (270 MHz, DMSO-d6) δ
(calcd for C18H19N5O2 337.1538); HPLC (method 2) 99.2% (tR
15.15 min). Anal. (C18H19N5O2·0.75CF3COOH·H2O) C, H, N.
=
12.41 (s, 1H), 11.85 (br s), 8.67 (s, 1H), 7.62 (d, J = 5.4 Hz, 1H), 7.58
(br s, 1H), 7.47 (dd, J = 3.0, 5.1 Hz, 1H), 7.25 (dd, J = 1.1, 5.1 Hz,
1H), 7.02 (s, 1H), 6.89 (s, 1H), 1.44 (s 9H); 13C NMR (150 MHz,
DMSO-d6) 157.8, 156.7, 140.9, 136.1, 134.8, 130.1, 128.9, 128.6,
125.2, 107.7, 105.6, 32.4, 31.0; HRMS (EI) m/z 342.1153 (M+) (calcd
for C17H18N4O2S 342.1150); HPLC (method 1) 96.3% (tR = 15.20
min). Anal. (C17H18N4O2S·0.75CF3COOH·0.5H2O) C, H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(3-pyridyl)methylidene]-2,5-piperazinedione (57). Yield of
29% from 9; mp 198−200 °C; IR (KBr, cm−1) 3422, 3189, 3124,
3093, 3045, 2886, 1697, 1677, 1661, 1420, 1396; 1H NMR (270 MHz,
CDCl3) δ 12.58 (s, 1H), 11.42 (br s, 1H), 8.78 (br s, 1H), 8.68−8.80
(m, 1H), 8.49−8.68 (m, 1H), 7.83 (d, J = 7.3 Hz, 1H), 7.54 (s, 1H),
7.41−7.52 (m, 1H), 7.08 (s 1H), 6.88 (s, 1H), 1.44 (s, 9H); 13C NMR
(150 MHz, DMSO-d6) 158.2, 156.2, 146.7, 144.5, 141.4, 141.1, 134.9,
130.2, 125.7, 124.6, 108.7, 105.9, 32.4, 31.0; HRMS (EI) m/z
337.1534 (M+) (calcd for C18H19N5O2 337.1538); HPLC (method 2)
96.1% (tR = 15.46 min). Anal. (C18H19N5O2·3CF3COOH·1.25H2O)
C, H, N.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(3-thienyl)methylidene]-2,5-piperazinedione (64). Yield of
39% from 9; mp 271−273 °C; IR (KBr, cm−1) 3166, 3099, 3030,
1
2974, 2874, 1686, 1646, 1390; H NMR (300 MHz, DMSO-d6) δ
12.41 (s, 1H), 11.81 (br s, 1H), 8.59 (s, 1H), 7.57 (br s, 2H), 7.46 (dd,
J = 2.4, 4.3 Hz, 1H), 7.25 (dd, J = 0.8, 4.6 Hz, 1H), 7.02 (s, 1H), 6.89
(s, 1H), 1.44 (s, 9H); 13C NMR (150 MHz, DMSO-d6) 157.8, 157.2,
140.6, 134.7, 134.3, 129.3, 127.1, 127.0, 125.6, 109.6, 104.8, 32.3, 30.9;
HRMS (EI) m/z 342.1154 (M+) (calcd for C17H18N4O2S 342.1150);
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-(4-pyridyl)methylidene]-2,5-piperazinedione (58). Yield of
50% from 9; mp 195−197 °C; IR (KBr, cm−1) 3463, 3182, 3055,
HPLC (method 1) 98.9% (tR
= 14.52 min). Anal.
1
2886, 1692, 1676, 1633, 1399; H NMR (300 MHz, DMSO-d6) δ
(C17H18N4O2S·CF3COOH·0.5H2O) C, H, N.
12.48 (br s, 1H), 12.42 (s, 1H), 10.87 (s, 1H), 8.78 (d, J = 5.7 Hz,
2H), 7.94 (d, J = 5.7 Hz, 2H), 7.93 (s, 1H), 6.93 (s, 1H), 6.77 (s, 1H),
1.39 (s, 9H); 13C NMR (150 MHz, DMSO-d6) 158.3, 155.6, 143.4,
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-
1-cyclohexylmethylidene]-2,5- piperazinedione (65). Yield of
31% from 9; mp 228−230 °C (dec); IR (KBr, cm−1) 3201, 3133, 2928,
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dx.doi.org/10.1021/jm2009088 | J. Med. Chem. 2012, 55, 1056−1071

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Article
2855, 1682, 1647, 1393; 1H NMR (300 MHz, DMSO-d6) δ 12.59 (br
s, 1H), 11.78 (br s, 1H), 10.30 (s, 1H), 8.05 (s, 1H), 6.74 (s, 1H), 5.69
(d, J = 10.2 Hz, 1H), 2.62−2.77 (m, 1H), 1.54−1.71 (m, 5H), 1.36 (s,
9H), 1.01−1.42 (m, 5H); 13C NMR (150 MHz, DMSO-d6) 157.8,
156.9, 140.1, 134.5, 126.1, 123.9, 79.6, 33.5, 32.3, 32.3, 30.9, 25.8, 25.4;
HRMS (EI) m/z 342.2053 (M+) (calcd for C19H26N4O2 342.2056);
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis of imidazole aldehydes, Table S1, Figures S1−S10,
1H and 13C NMR spectra, HPLC charts, crystallographic
information files (Tables S2 and S3), and elemental analysis
table. This material is available free of charge via the Internet at
http://pubs.acs.org.
HPLC (method 1) 99.4% (tR
(C19H26N4O2·CF3COOH·H2O) C, H, N.
= 16.72 min). Anal.
Tubulin Binding Assay. Fluorescence spectra were measured at
37 °C as described previously.29,31 Bovine or porcine tubulin (0.5 μM)
in MES buffer (0.1 M MES, 0.5 mM MgCl2, 1 mM EGTA, 1 mM
GTP, pH 6.8) was incubated with different concentrations of the test
compounds (0−12 μM, 1% DMSO) at 37 °C for 1 h. After incubation,
the fluorescence of each tubulin sample was measured (excitation at
290 nm, emission at 340 nm) using a spectrofluorometer (TECAN
Spectra Fluor Plus).
Cytotoxic Activity Assay against HT-29 Cells and HuVEC-
s.19a,32 HT-29 cells were purchased from ATCC and were maintained
in McCoy’s 5A medium containing 10% fetal bovine serum
supplemented with 1% penicillin/streptomycin at 37 °C in a
humidified 5% CO2 atmosphere. HuVECs were obtained from
Cambrex Bio Science (Walkersville, MD), used between passages 2
and 6, and grown in EGM-2 medium at 37 °C in a humidified 5% CO2
atmosphere. For the growth inhibition assays, cells were plated in 96-
well plates at an appropriate density the day before compound
addition. Stock solutions of compounds were prepared in DMSO.
Serially diluted compounds were added to the cells, resulting in a final
concentration range from 20 μM to 2 pM. Fourty-eight hours later, 10
μL of 0.2 mg/mL resazulin solution in PBS buffer was added to each
well, and the cells were incubated for an additional 3−6 h. The
fluorescence of the reduction product of resazulin was measured using
a Fusion microplate fluorometer (Packard Bioscience) with λex = 535
nm and λem =590 nm filters.
AUTHOR INFORMATION
■
Corresponding Author
*Tel/fax: +81-42-676-3275. E-mail: yhayashi@toyaku.ac.jp.
ACKNOWLEDGMENTS
■
This research was supported by grants from MEXT (Ministry
of Education, Culture, Sports, Science and Technology), Japan,
including the Grant-in Aid for Young Scientists (B) 21790118
and the Grant-in Aid for Scientific Research (B) 20390036, and
also in part by the Frontier Research Program of the Ministry of
Education, Science and Culture of Japan and grants from the
Ministry of Education, Science and Culture of Japan and Japan
Health Science Foundation. The authors acknowledge Ms. M.
Adachi, Mr. Y. Shimada, Mr. Y. Mori, Ms. Y. Kido and Mr. Y.
Yamamoto for their assistance in the synthesis of derivatives.
We also thank Ms. C. Sakuma and Mr. H. Fukaya of the Tokyo
University of Pharmacy and Life Sciences for mass spectra,
NMR, and crystallographic measurements.
ABBREVIATIONS
■
ATCC, American type culture collection; BSA, bovine serum
albumin; CoMFA, comparative molecular field analysis;
Cs2CO3, cesium carbonate; DAPI, 4′,6-diamidino-2-phenyl-
indole; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DKP, dike-
topiperazine; 3D-QSAR, three-dimensional quantitative struc-
ture−activity relationship; DU 145, human prostate carcinoma
cell lines; EGTA, ethylene glycol tetraacetic acid; EI, electron
ionization; ESI, electrospray ionization; FITC, fluorescein
isothiocyanate; HPLC, high-performance liquid chromatogra-
phy; HRMS, high-resolution mass spectrometry; HT-29,
human colon adenocarcinoma cell lines; HuVECs, human
umbilical vein endothelial cells; IC50, 50% inhibitory concen-
tration; Ka, association constant; Kd, dissociation constant;
LiAlH4, lithium aluminum hydride; MAPs, microtubule
associated proteins; MDR, multidrug resistance; MES, 2-(N-
morpholino)ethanesulfonic acid; NSCLC, non-small-cell lung
carcinoma; ODS, octadecylsilyl; ORTEP, Oak Ridge thermal
ellipsoid plot; PBS, phosphate-buffered saline; SAR, structure−
activity relationship; SEM, standard error of the mean; TFA,
trifluoroacetic acid; TLC, thin-layer chromatgraphy; TMS,
tetramethylsilane; USAN, United States adopted name; VDA,
vascular disrupting agent
Preparation of Tubulin and Microtubule Polymerization
Assay in Vitro. Tubulin except for MAPs was purified from bovine
brain through the two cycles of the polymerization−depolymerization
method described as previously.33 Turbility assays of tubulin were
performed by incubating 1 mg/mL tubulin in RB buffer (100 mM
MES, 1 mM EGTA, 0.5 mM MgCl2, pH6.8) with 1 mM GTP and 1 M
glutamate. Increase of absorbance at 350 nm was monitored in
cuvettes at 37 °C using a thermostatic spectrophotometer (Beckman
Coulter Inc., Brea, CA)
Analysis of Cell Cycle Progression. HeLa cells (1 × 105 cells/
mL) were treated with various concentrations of each compound for
18 h and then harvested and stained with propidium iodide solution.
DNA contents were measured by flow cytometric analysis (PARTEC
CyFlow PA; Partec GmbH, Munster, Germany). Cell populations of
each cell cycle phase were calculated using Multi Cycle AV
(PhoenixFlow Systems).
Microtubule Polymerization Assay on HuVECs. Microtubule
depolymerization assay was performed as described previously.19a
HuVECs were plated onto sterile tissue culture treated coverslips
(Fisher, Hampton, NH) in six-well plates. Following overnight
incubation the proliferating HuVECs were treated with test
compounds or vehicle [0.25% (v/v) DMSO]. The plates were
returned to the incubator for 30 min. Following fixation in 10% (v/v)
neutral buffered formalin at room temperature, the cells were
permeabilized in 0.2% (v/v) Triton X-100/dPBS before transferring
to a humidified chamber and blocking for 2 h in antibody buffer [2%
(w/v) BSA/0.1% (v/v) Tween-20/dPBS]. The coverslips were
incubated with 0.1 mg/mL mouse anti-α-tubulin in antibody buffer
for 1 h before washing and incubation with 1 mg/mL goat antimouse−
FITC for 1 h in the dark. Finally, to visualize the nuclei, the cells were
washed and treated with 2 mg/mL DAPI before mounting with
Vectashield mounting media. The cells were imaged using a 60× oil
immersion objective on an upright microscope (Olympus BX51). The
images were digitally captured using a CCD camera and Magnafire 2.0
software (Olympus, Melville, NY).
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