[4-(Imidazol-1-yl)thiazol-2-yl]phenylamines. A novel class of highly potent colchicine site binding tubulin inhibitors: Synthesis and cytotoxic activity on selected human cancer cell lines

Article abstract of DOI:10.1021/jm060545p

Synthesis and cytotoxic activity in the submicromolar range of a series of [4-(imidazol-1-yl)thiazol-2-yl]-phenylamines are described. Cell cycle dependent cytotoxicity on RKO human colon carcinoma cells with inducible expression of p27^kip1 and the influence on microtubule formation were investigated. Considering the significant correlation between the IC₅₀ values of tubulin polymerization inhibition, [³H]colchicine competition, and cytotoxicity of the investigated compounds, tubulin is the main cellular target. The inhibition of microtubule formation was shown to be mediated by interference with the colchicine binding site of tubulin. In depth analysis of the investigated compounds allowed the identification of modifications that altered the pharmacological profile of the compounds from a mitosis-inducing phenotype to a G1 cell cycle arresting phenotype.

Full text of DOI:10.1021/jm060545p

J. Med. Chem. 2006, 49, 5769-5776
5769
[4-(Imidazol-1-yl)thiazol-2-yl]phenylamines. A Novel Class of Highly Potent Colchicine Site
Binding Tubulin Inhibitors: Synthesis and Cytotoxic Activity on Selected Human Cancer Cell
Lines
Siavosh Mahboobi,*,† Andreas Sellmer,† Heymo Ho¨cher,† Emerich Eichhorn,† Thomas Ba¨r,‡ Mathias Schmidt,‡ Thomas Maier,‡
Josef F. Stadlwieser,§ and Thomas L. Beckers*,‡
Department of Pharmaceutical Chemistry I, UniVersity of Regensburg, D-93040 Regensburg, Germany, and Therapeutic Area Oncology and
Enabling Technologies, ALTANA Pharma AG, D-78467 Konstanz, Germany
ReceiVed May 10, 2006
Synthesis and cytotoxic activity in the submicromolar range of a series of [4-(imidazol-1-yl)thiazol-2-yl]-
phenylamines are described. Cell cycle dependent cytotoxicity on RKO human colon carcinoma cells with
inducible expression of p27kip1 and the influence on microtubule formation were investigated. Considering
the significant correlation between the IC50 values of tubulin polymerization inhibition, [3H]colchicine
competition, and cytotoxicity of the investigated compounds, tubulin is the main cellular target. The inhibition
of microtubule formation was shown to be mediated by interference with the colchicine binding site of
tubulin. In depth analysis of the investigated compounds allowed the identification of modifications that
altered the pharmacological profile of the compounds from a mitosis-inducing phenotype to a G1 cell cycle
arresting phenotype.
Introduction
Within the past decade our understanding of malignant cell
growth and the regulation of the cell cycle machinery has offered
several new opportunities for targeted cancer therapy and the
promise of a broader therapeutic window. Within mitosis, Râ-
tubulin heterodimers building up the mitotic spindle still
Figure 1. Structure of lead compound 4-bromo[4-(2,4-dimethylthiazol-
5-yl)thiazol-2-yl]phenylamine (1) and general structure of [4-(imidazol-
1-yl)thiazol-2-yl]phenylamines (2).
represent an attractive target for the development of anticancer
drugs. Various natural, semisynthetic or fully synthetic new
tubulin inhibitors are currently in clinical development like
azaepothilone (ixabepilone, Bristol-Myers Squibb) in advanced
phase III clinical studies.1 Another approach in targeted cancer
therapy is represented by small-molecule inhibitors of cyclin-
dependent kinases (CDKsa) having a key function in cell cycle
regulation.2 It is a feature of most cancer cells that they harbor
a deregulation of at least one CDK function, making CDK
inhibitors particularly attractive as potential therapeutic agents.2-4
In the course of our search for new antimitotic compounds,
we identified compound 1 (Figure 1) from the in-house
compound archive as a promising lead. While evaluating the
chemical space around this compound, we also investigated
structurally related imidazoles of substructure 2 (Figure 1).
Here, we report on the structural refinement of compound 2,
which led to a novel series of highly active antimitotic, tubulin
interfering agents with a [4-(imidazol-1-yl)thiazol-2-yl]pheny-
lamine type structure, inhibiting microtubule polymerization by
interfering with the colchicine binding site of â-tubulin.
We further report on analogues with an altered cellular profile
shifting from an antimitotic phenotype to a G1 cell cycle arrest-
ing phenotype within this series. These compounds were identi-
fied during the process of lead optimization by showing distinct
new phenotypic alterations in cancer cells. While antimitotic
drugs typically induce a rounding of cells, indicative of mitotic
accumulation before the onset of apoptosis, these compounds
induced long cell body protrusions typical for inducible expres-
sion of CDK inhibitors such as p21Waf1 or p27Kip1
.
Results and Discussion
Chemistry. According to Scheme 1, the desired [4-(imidazol-
1-yl)thiazol-2-yl]phenylamines were prepared from commer-
cially accessible anilines (3) employing the strategy of Ras-
mussen,5 which includes formation of N-aryl-N′-benzoylthioureas
(4) and debenzoylation to the respective arylthioureas (5). After
ring closure with ethyl bromoacetate, chlorination of the
resulting 2-phenylaminothiazol-4-ones (6) with phosphorus
oxychloride analogous to Brown et al.6 yields (4-chlorothiazol-
2-yl)phenylamines (7) as intermediates. The desired [4-(imida-
zol-1-yl)thiazol-2-yl]phenylamines (8) were obtained by nu-
cleophilic aromatic substitution in DMF solution, using the
respective imidazoles in excess as reagents and bases.
To prepare the corresponding sulfonamides (Scheme 2), a
modified synthetic strategy to obtain the 2-phenylaminothiazolin-
4-one intermediates 6 was explored. Alkylation of 2-thioxothiazo-
lidin-4-one (13) with methyl iodide in aqueous NaOH according
to Khodair led to 2-methylsulfanylthiazol-4-one (14).7 The de-
sired 4-(4-oxo-4,5-dihydrothiazol-2-ylamino)benzenesulfona-
mide (6j) was obtained by nucleophilic displacement at C-2 of
* To whom correspondence should be addressed. For S.M.: phone, (+49)
(0) 941-9434824; fax, (+49) (0) 941-9431737; e-mail, siavosh.mahboobi@
chemie.uni-regensburg.de. For T.L.B.: phone, (+49) (0) 7531 842974; fax,
(+49) (0) 7531 8492974; e-mail, Thomas.Beckers@altanapharma.com.
† University of Regensburg.
‡ Therapeutic Area Oncology, ALTANA Pharma AG.
§ Enabling Technologies, ALTANA Pharma AG.
a Abbreviations: CDK, cyclin dependent kinase; G, gap; M, mitosis;
MAP, microtubule-associated protein(s); FCS, fetal calf serum; DMEM,
Dulbecco’s modified Eagle’s medium; PBS, phosphate buffered saline;
FACS, fluorescence-activated cell sorting.
10.1021/jm060545p CCC: $33.50 © 2006 American Chemical Society
Published on Web 08/29/2006

5770 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
Mahboobi et al.
Scheme 1. Preparation of [4-(imidazol-1-yl)thiazol-2-yl]phenylamines
Scheme 2. Preparation of [4-(imidazol-1-yl)thiazol-2-ylamino]phenylbenzenesulfonamides
14 by sulfanilamide.8 Treatment with phosphorus oxychloride
and SN reaction as described above yielded the desired 4-[4-
(imidazol-1-yl)thiazol-2-ylamino]benzenesulfonamides.
Biological Results. Tubulin inhibitors interfere with the M
phase of the cell cycle by altering the dynamics of tubulin
polymerization, which finally results in the induction of apo-

Phenylamines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5771
Figure 2. Cell cycle dependent antimitotic activity of 9d: (A) determination of the cytotoxicity after 72 h on proliferating (filled triangles) versus
arrested (open boxes) RKOp27 cells as described in Experimental Section; (B) methylene blue/eosin staining of proliferating (left) and arrested
(right) RKOp27 cells after 24 h of treatment with 10 µM 9d; (C) cell cycle distribution after treatment with 10 µM 9d, where control cells (left)
were treated with DMSO; 24 h later the cells were harvested, fixed, and stained with propidium iodide as described in Experimental Section prior
to analysis by flow cytometry; (D) interference of 9d with MAP-containing bovine brain tubulin polymerization. The assay was conducted as
described in Experimental Section.
Table 1. Structures of 1 and Imidazoles 8-10 and IC50 Values on Arrested versus Proliferating RKOp27 Cellsa
IC50 ( SD (µM)
compd
R1
R2
R3
RKOp27 proliferating
RKOp27 arrested
3H colchicine
1
8a
9a
10b
9b
10c
9c
0.7 ( 0.1
>100 ( 0
32.5 ( 2.5
0.4 ( 0.1
30 ( 0
0.25 ( 0.05
1.6 ( 0.4
>100 ( 0
0.2 ( 0.05
0.45 ( 0.15
49 ( 1
>100 ( 0
>100 ( 0
22.5 ( 7.5
22.5 ( 7.5
0.016
30 ( 0
>100 ( 0
>100 ( 0
45 ( 15
>100 ( 0
>100 ( 0
>100 ( 0
>100 ( 0
>100 ( 0
>100 ( 0
>100 ( 0
>100 ( 0
>100 ( 0
60 ( 0
1 ( 0
H
H
4-Br
H
H
Me
H
Me
H
H
Me
H
H
Me
H
H
Me
Me
Me
Me
Me
H
Me
Me
Me
Me
Me
Me
Me
4 ( 1
4-Br
4-OMe
4-OMe
4-OMe
4-Me
4-Me
4-SO2NH2
3-Br
3-Br
3-OMe
3-OMe
7.5 ( 1.5
8 ( 2
8c
10d
9d
9 g
10e
9e
10f
9f
colchicine
6.25 ( 0.75
0.75 ( 0.45
Me
H
>100 ( 0
>5
>0.5
0.5 ( 0.2
4
podophyllotoxin
0.029
a For selected compounds, the IC50 values for [3H]colchicine competition are also given. Values are the average of two independent experiments ( SD
conducted in triplicate (colchicine competition) or quadruplicate samples for each concentration.
ptosis within mitosis. For evaluation of a mitosis-dependent
mode of action, the compounds were tested in the genetically
engineered human colon carcinoma cell line RKOp27 with
ponasterone-A-inducible expression of the cell division cycle
of derivatives tested displayed a significant cell cycle dependent
cytotoxicity down to the low micromolar level on proliferating
cells and were up to 3000-fold less active in arrested cells. The
exemplary cell cycle-dependent activity of 9d is shown in Figure
2A. Proliferating cells were arrested in mitosis with randomly
separated condensed chromosomes (Figure 2B). Flow cytometric
analysis to measure the DNA content of proliferating cells
confirmed that the majority of cells were arrested in the G2/M
stage of the cell cycle (Figure 2C). Since â-tubulin is a prime
target for many compounds with antimitotic mode of action,
we investigated the potential interference with microtubule
kinase inhibitor p27kip1 9,10 Induction of p27kip1 arrests proliferat-
.
ing RKO cells in the G1 phase of the cell division cycle, thereby
inducing complete resistance to antimitotic agents such as
paclitaxel as a stabilizing compound or vincristine as destabiliz-
ing tubulin inhibitor.1
We utilized the RKOp27 system to test the cell cycle depen-
dent cytotoxicity of the compounds described herein. A number

5772 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
Mahboobi et al.
Table 2. Structures of Benzimidazoles 11 and 12 and Their IC50 Values
be mandatory. This is demonstrated in compound 9a, in which
a methyl group in the R3 position leads to moderate activity
compared to compound 8a, which is unsubstituted and com-
pletely inactive. A methyl group in the R2 position may turn
moderate activity into submicromolar activity, as shown for
compound 10b, which is 60-fold more active than compound
9b. The methoxy derivative 10c represents the most active
compound in these series, exhibiting an IC50 of 0.2 µM.
Compound 10b compares directly to compound 1, demonstrating
the superiority of the imidazole series versus the compounds
with a terminal thiazole moiety.
Recognizing that R2 ) CH3 at the imidazole ring is beneficial,
we also evaluated related benzimidazoles. Annellation generally
leads to an increase in activity, as shown in Table 2. The
4-bromo compound 12b, with an IC50 of 0.02 µM, is 20-fold
more active than the corresponding imidazole 10b. As in the
imidazole series, a substituent in para position of the phenyl
moiety is mandatory, while meta substituents are not tolerated.
In close analogy to the imidazole series, the removal of the
methyl group (R2) leads to a complete loss of activity.
(Cytotoxicity) on Arrested versus Proliferating RKOp27 Cellsa
IC50 ( SD (µM)
RKOp27
RKOp27
arrested
compd
R1
R2 proliferating
3H colchicine
11a
12a
12c
11c
12f
12b
11b
12e
12g
H
H
4-OMe
4-OMe
3-OMe
4-Br
4-Br
3-Br
4-SO2NH2 Me
H
Me
>100 ( 0
40 ( 0
>100 ( 0
50 ( 0
Me 0.025 ( 0.005
H
Me
Me
H
52.5 ( 2.5 0.75 ( 0.05
>100 ( 0
>100 ( 0
40 ( 10
60 ( 0
0.09 ( 0.07
>100 ( 0
36 ( 14
60 ( 0
>100 ( 0
85 ( 5
1.25 ( 0.25
100 ( 0
Me
45 ( 5
95 ( 5
a For selected compounds, the IC50 values for [3H]colchicine competition
are also given. Values are the average of two independent experiments (
SD conducted in triplicate (colchicine competition) or quadruplicate samples
for each concentration.
To further elucidate the mechanism of interference with
microtubule formation, we examined whether selected [4-(imi-
dazol-1-yl)thiazol-2-yl]phenylamines directly bind to â-tubulin.
A competition binding assay was employed in which the binding
of [3H]colchicine to biotinylated bovine brain tubulin is quanti-
fied.11 As depicted in Figure 3B, 10b is able to bind to the
colchicine binding site of â-tubulin with an IC50 value of
approximately 3 µM. As summarized in Tables 1 and 2, all
compounds with potent cell cycle dependent cytotoxicity also
interfered with tubulin by binding to the colchicine site of
tubulin. Known colchicine competitive tubulin binders such as
podophyllotoxin12 or the 2-aroylindole D-6413113 interfere in
this assay with IC50 values of 4 and 0.51 µM, respectively.
polymerization. 9d inhibited the in vitro polymerization of
MAP-containing bovine brain tubulin with an IC50 of ap-
proximately 1 µM (Figure 2D).
The respective IC50 values of further investigated [4-(imida-
zol-1-yl)thiazol-2-yl]phenylamines on arrested versus proliferat-
ing cells are summarized in Table 1, again emphasizing the
profound cell cycle dependent cytotoxic activity.
These IC50 data indicate that minor changes in the substitution
pattern have a dramatic impact on potency. It appears evident
that R1 inevitably has to be positioned in the para position. A
shift of the substituent to the meta position leads to a dramatic
loss of potency and to unspecific cytotoxicity. From the
representative results in Table 1, we assumed that electron-
donating groups on the phenyl moiety are beneficial, while
electron-withdrawing groups like sulfamoyl are detrimental. The
presence of methyl substituents on the imidazole turned out to
Inhibition of tubulin polymerization correlated well with
colchicine competition for compounds 9d (3 versus 0.75 µM),
12b (1.2 versus 1.25 µM), and 12c (1.25 versus 0.75 µM). The
antimitotic activity of 10b was determined by its cell cycle
dependent cytotoxicity (Figure 3A) together with its ability to
Figure 3. Competition of [3H]colchicine binding to â-tubulin by 10b. (A) The cell cycle dependent cytotoxicity was determined as described in
Figure 2. (B) Competition of [3H]colchicine binding to biotinylated â-tubulin. Various concentrations of 10b (as indicated) were added, and the
IC50 was calculated using Graph Pad Prism software. (C) Methylene blue/eosin staining of RKOp27 cells after 24 h of treatment with 5 µM 10b.
Note the occurrence of a large amount of cells arrested in mitosis. (D) Cell cycle distribution after treatment with 10 µM 9d. Control cells (left)
were treated with DMSO. Treatment was conducted exactly as described in Figure 2 except that A549 human non-small-cell lung cancer cells were
used instead of RKOp27 cells. See Experimental Section and the caption of Figure 2 for details.

Phenylamines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5773
Figure 4. G1 cell cycle arresting properties of 9b. (A) The cell cycle dependent cytotoxicity was determined as described in Figure 2. (B) Micrographs
of RKOp27 cells treated for 72 h with 30 µM 9b (lower left panel). Control cells (upper left panel) were treated with DMSO. Methylene blue/eosin
staining of RKOp27 cells was conducted with cells treated for 24 h with 30 µM 9b (lower right panel). Control cells were treated with DMSO
(upper right panel). (C) Cell cycle distribution after treatment with 30 µM 9b. The cells were seeded out and treated as indicated in part B. Control
cells (upper panel) were treated with DMSO. Twenty-four hours later, the cells were harvested, fixed, and stained with propidium iodide as described
in Experimental Section prior to analysis by flow cytometry.
polymerization in addition to its G1 arresting capacity. Flow
cytometry analyses (Figure 4C) to determine the DNA content
confirmed that most of the cells have been arrested in the G1
stage of the cell cycle rather than in mitosis.
Byth et. al.3,14 previously published a SAR study on several
[4-(imidazo[1,2-a]pyridine-3-yl)pyrimidin-2-yl]phenylamines as
inhibitors of CDK2. In this publication, 3-chloro[4-(benzimi-
dazol-1-yl)pyrimidin-2-yl]phenylamine (15) is reported to be a
CDK2 inhibitor.
Since bioisosterism15 between 2-aminopyrimidin and 2-ami-
nothiazole scaffolds is well-known in the field of kinase
inhibitors, exemplified by imatinib (16)16 and its thiazolyl
analogue 1717,18 (Figure 5), which are both known as c-Kit
inhibitors, one may hypothesize that the G1 cell cycle arresting
profile of 9b is related to CDK2 inhibition. Further investigations
will be necessary to substantiate the hypothesis, whether small
chemical modifications can shift the properties from an opti-
Figure 5. Chemical structures of 3-chloro[4-(benzimidazol-1-yl)-
pyrimidin-2-yl]phenylamine (15), a potent CDK2 inhibitor, imatinib
(16), and its bioisosteric thiazolyl analogue.
mized microtubule interfering agent to a cell cycle antagonist
by inhibition of cyclin-dependent kinase activity or by different
mechanistic interactions with the cell cycle machinery.
arrest RKOp27 cells with a large proportion of cells displaying
Conclusion
condensed chromosomes (Figure 3C). Cell cycle analyses by
We describe a novel series of antimitotic, microtubule
flow cytometry confirmed that the majority of the treated cells
destabilizing, colchicine-competitive compounds. Starting from
were arrested in the G2/M phase (Figure 3D)
a lead identified by high-throughput screening, we were able
Interestingly, small modifications changed the cellular profile
to rapidly synthesize compounds of a related scaffold, exhibiting
from a mitosis-confined phenotype to a G1 cell cycle arresting
cellular activities in the lower nanomolar range. A set of highly
profile. This is exemplified by 9b (Figure 4), which displayed
active as well as inactive compounds, with only minor structural
cell cycle dependent cytotoxicity on RKOp27 cells. However,
differences, allowed us to establish a compelling structure-
the potency was much less pronounced and a plateau in viability
activity relationship. Furthermore, we surprisingly found that
was reached at about 50% of control cells after 72 h of treatment
minor structural modifications in the investigated inhibitors may
(Figure 4A). Morphological analysis did not show signs of
also shift the properties from an optimized microtubule interfer-
cytotoxicity (Figure 4B, lower left panel). Moreover, an
ing agent toward a G1 cell cycle arresting compound.
intriguing change in the morphological shape of RKOp27 cells
was observed with the generation of cell body protrusions
(indicated by arrows). These morphological alterations were also
Experimental Section
Biological Methods. Microtubule Polymerization Assay. The
observed upon inducible expression of cell cycle inhibitors such
assay was performed as described.19 For details, see Supporting
as p21Waf1 or p27Kip1 or upon treatment with compounds of CDK
Information.
inhibitory capacity (data not shown). Similar observations were
Cell Viability Assay. Metabolic activity correlating with cell
made with 9a and 9f. Methylene blue/eosin staining of treated
proliferation/viability was quantified by the Alamar Blue assay as
versus control cells illustrated a slightly increased mitotic index
of 8.5%. M-phase cells often displayed abnormal mitosis
characterized by condensed chromosomal material that was
randomly distributed within the cells. Therefore, it cannot be
ruled out that 9b is still able to interfere with microtubule
described20 using RKOp27 colon carcinoma cell lines.9 For details,
see Supporting Information.
Flow Cytometric Analysis. After treatment with test compound,
cells were harvested by trypsinization, and an aliquot of 1 × 106
cells was washed once with cold PBS and then fixed with cold

5774 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
Mahboobi et al.
70% ethanol. The DNA was stained with a solution containing 25
µg/mL propidium iodide and 10 µg/mL RNase A in PBS for 6 h.
Cell cycle distribution was analyzed with a FACScan flow
cytometer (Becton Dickinson, San Jose, CA) at an excitation
wavelength of 488 nm.
Cell Staining. Upon completion of compound treatment, cells
were harvested by trypsinization. An aliquot (5 × 103 cells) was
spun onto glass slides using a cytospin and subsequently fixed and
stained with the Hemacolor kit (Merck Darmstadt, Germany)
according to the manufacturer’s instructions.
d6): δ (ppm) ) 11.58 (s, 1H), 8.37-7.47 (m, br, 1H), 7.33 (s,
2H), 7.21-6.87 (m, br, 1H), 4.00 (s, 2H). Anal. (C9H7BrN2OS) C,
H, N.
(3-Methoxyphenylamino)thiazol-4-one (6f).26 6f was prepared
from 3-methoxyphenylthiourea (5f) as described above. Yield: 9.37
1
g (77%), beige crystals. H NMR (DMSO-d6): δ (ppm) ) 11.73
(s, 0.5 × 1H), 11.13 (s, 0.5 × 1H), 7.39-7.20 (m, br, 2H), 6.72 (s,
br, 1H), 5.57-6.52 (m, br, 1H), 4.00 (s, 0.5 × 2H), 3.98 (s, 0.5 ×
2H), 3.70 (s, 3H).
4-(4-Oxo-4,5-dihydrothiazol-2-ylamino)benzenesulfonamide
(6g) was prepared from 147 according to the literature.8 Yield: 6.50
g (73%), colorless crystals. Mp: 240 °C.27 1H NMR (DMSO-d6):
δ (ppm) ) 11.75 (s, 1H, exchangeable), 7.82 (s, br, 3H), 7.32 (s,-
2H exchangeable), 7.10 (s, br, 1H), 4.03 (s, 2H).
(4-Chlorothiazol-2-yl)arylamines 7a-g. General Procedures.
Procedure A. A mixture of the appropriate arylaminothiazol-4-
one (4.8 mmol), 3 mL of POCl3, and 0.4 mL of pyridine was
refluxed for 3 h. After the mixture was cooled to room temperature,
the solvent was removed under reduced pressure. The residue was
dissolved in ether, and the solution was washed twice with 5%
aqueous NaOH, brine, and water. The solvent was dried (Na2SO4)
and removed under reduced pressure. The crude product was
purified by column chromatography.
Procedure B. A mixture of the appropriate arylaminothiazol-
4-one (24 mmol), tetrabutylammonium bromide (60 mmol), di-
methylanilin (24 mmol), and POCl3 (144 mmol) in 250 mL of
acetonitrile was stirred 5 h at 80 °C. To remove the excess of POCl3,
water was added carefully at 0 °C. Then an amount of 100 mL of
dichloromethane was added, and the organic layer was separated,
washed with brine and water, and dried (Na2SO4). The solvent was
removed under reduced pressure. Purification by column chroma-
tography led to colorless crystrals.
Colchicine Competition Binding Assay. The assay was con-
ducted basically as described.11 For details, see Supporting Informa-
tion.
Chemical Procedures. General. NMR spectra were recorded
with a Bruker Avance 300 MHz spectrometer at 300 K, using TMS
as an internal standard. IR spectra (KBr or pure solid) were
measured with a Bruker Tensor 27 spectrometer. Melting points
were determined with a Bu¨chi B-545. MS spectra were measured
with a Finnigan MAT 95 (EI, 70 eV) with a MAT 710A (CI-MS,
NH3). All reactions were carried out under nitrogen atmosphere.
Elemental analyses were performed by the Analytical Laboratory
of the University of Regensburg.
4-Bromo[4-(2,4-dimethylthiazol-5-yl)thiazol-2-yl]phenyl-
amine (1). Compound 1 is commercially available at Ambinter
Stock Screening Collection, Ambinter, 50 Avenue de Versailles,
Paris, F-75016, France; CAS No. 315705-49-8.
Arylthioureas 5b-f were prepared from the corresponding
anilines according to the excellent method described by Rasmussen5
by debenzoylation of the respective N-aryl-N′-benzoylthioureas 4b-
f.
Arylaminothiazol-4-ones 6a-f. General Procedure. The aryl-
thioureas 5a-f (120 mmol) were dissolved in 400 mL of ethanol
pa at 50 °C. To this solution ethyl bromoacetate (156 mmol) was
added, and the mixture was stirred at this temperature for 2 h. After
cooling to room temperature, the mixture was neutralized with
aqueous ammonia. Dropwise addition of water when stirring led
to precipitation of the product, which was removed by filtration
and dried. Trituration of the resulting solid with petroleum ether
(40/60) affords yellow crystals. The title compounds exist as a
mixture of tautomerics21 in DMSO solution. Therefore, the signals
in the 1H NMR spectra are given as observed either in the form of
the resuling coalescence spectra or by indicating the proportion in
signal intensity for every observable signal.
(4-Chlorothiazol-2-yl)phenylamine (7a). 7a was prepared from
phenylaminothiazol-4-one (6a) according to procedure A. Yield:
1
3.35 g (32%), colorless crystals. Mp: 96.8-97.4 °C. H NMR
(DMSO-d6): δ (ppm) ) 10.46 (s, 1H), 7.54 (d, 2H), 7.34 (t, 2H),
7.02 (t, 1H), 6.85 (s, 1H). Anal. (C9H7ClN2S) C, H, N.
(4-Chlorothiazol-2-yl)-4-bromophenylamine (7b). 7b was pre-
pared from 4-bromophenylaminothiazol-4-one (6b) according to
procedure B. Yield: 0.22 g (42%), colorless crystals. Mp: 175
°C. 1H NMR (DMSO-d6): δ (ppm) ) 10.60 (s, 1H), 7.53 (m, 4H),
6.90 (s, 1H). Anal. (C9H6BrClN2S) C, H, N.
(4-Chlorothiazol-2-yl)-4-methoxyphenylamine (7c). 7c was
prepared from 4-methoxyphenylaminothiazol-4-one (6c) according
to procedure B. Yield: 0.90 g (27%), colorless crystals. Mp: 105
°C. 1H NMR (DMSO-d6): δ (ppm) ) 10.25 (s, 1H), 7.46 (d, 2H),
6.93 (d, 2H), 6.78 (s, 1H), 3.72 (s, 3H). Anal. (C10H9ClN2OS) C,
H, N.
(2-Phenylamino)thiazol-4-one (6a).22 6a was prepared from
commercially available phenylthiourea (Acros) as described above.
1
Yield: 5.65 g (82%), yellow crystals. Mp: 178.4-178.5 °C. H
NMR (DMSO-d6): δ (ppm) ) 11.73 (s, 0.5 × 1H), 11.15 (s, 0.5
× 1H), 7.70 (d, 0.5 × 2H, J ) 7.4 Hz), 7.40-7.37 (m, 2H), 7.18-
7.13 (m, 1H), 7.0 (d, 0.5 × 2H), 4.01 (s, 0.5 × 2H), 3.97 (s, 0.5 ×
2H).
(4-Chlorothiazol-2-yl)-p-tolylamine (7d). 7d was prepared from
p-tolylaminothiazol-4-one (6d) according to procedure B. Yield:
(4-Bromophenylamino)thiazol-4-one (6b).23 6b was prepared
from 4-bromophenylthiourea (5b) as described above. Yield: 7.90
g (67%), beige crystals. Mp: 229-230 °C.24 1H NMR (DMSO-
d6): δ (ppm) ) 11.83 (s, 0.5 × 1H), 11.28 (s, 0.5 × 1H), 7.80-
7.72 (m, br, 1H), 7.62-7.50 (m, br, 2H), 6.95-6.90 (m, br, 1H),
4.08 (s, 0.5 × 2H), 4.00 (s, 0.5 × 2H).
1.05 g (44%), beige crystals. Mp: 114.5 °C. H NMR (DMSO-
1
d6): δ (ppm) ) 10.38 (s, 1H), 7.58 (d, 2H), 7.17 (d, 2H), 6.80 (s,
1H), 2.25 (s, 3H). Anal. (C10H9ClN2S) C, H, N.
(4-Chlorothiazol-2-yl)-3-bromophenylamine (7e). 7e was pre-
pared from 3-bromophenylaminothiazol-4-one (6e) according to
procedure B. Yield: 1.32 g (62%), colorless crystals. Mp: 133
°C. 1H NMR (DMSO-d6): δ (ppm) ) 10.66 (s, 1H), 7.90 (s, 1H),
7.46 (d, 1H), 7.29 (t, 1H), 7.16 (d, 1H), 6.93 (s, 1H). Anal. (C9H6-
BrClN2S) C, H, N.
(4-Methoxyphenylamino)thiazol-4-one (6c).25 6c was prepared
from 4-methoxyphenylthiourea (5c) as described above. Yield:
1
21.45 g (88%), brown crystals. Mp: 178.1-178.2 °C. H NMR
(DMSO-d6): δ (ppm) ) 11.20 (s, 1H), 7.60 (d, 1H), 6.97-6.93
(m, 3H), 3.96 (s, 0.5 × 2H), 3.89 (s, 0.5 × 2H), 3.75 (s, 3H).
(4-Chlorothiazol-2-yl)-3-methoxyphenylamine (7f). 7f was
prepared from 3-methoxyphenylaminothiazol-4-one (6f) according
to procedure A. Yield: 1.60 g (21%), colorless crystals. Mp: 86-
(p-Tolylamino)thiazol-4-one (6d).26 6d was prepared from
p-tolylthiourea (6d) as described above. Yield: 19.85 g (80%),
1
87 °C. H NMR (DMSO-d6): δ (ppm) ) 10.47 (s, 1H), 7.26-
1
yellow crystals. Mp: 179.2-179.3 °C. H NMR (DMSO-d6): δ
7.19 (m, 2H), 7.07 (d, 1H), 6.86 (s, 1H), 6.58 (d 1H). Anal.
(C10H9ClN2OS) C, H, N.
(ppm) ) 11.33 (s, 1H), 7.56 (d, 0.5 × 2H), 7.18 (d, 2H), 6.92 (d,
0.5 × 2H), 3.98 (s, 0.5 × 2H), 3.92 (s, 0.5 × 2H), 2.28 (s, 3H).
4-(4-Chlorothiazol-2-ylamino)benzenesulfonamide (7g). 7g
was prepared from 4-(4-oxo-4,5-dihydrothiazol-2-ylamino)benzene-
sulfonamide (6g) according to procedure B. Yield: 3.90 g (66%),
(3-Bromophenylamino)thiazol-4-one (6e). Preparation from
3-bromophenylthiourea (5e) as described above. Yield: 13.42 g
1
1
(63%), beige crystals. Mp: 177.7-177.8 °C. H NMR (DMSO-
colorless crystals. Mp: 165.5 °C (dec). H NMR (DMSO-d6): δ

Phenylamines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5775
(ppm) ) 10.88 (s, 1H, exchangeable), 7.78 (d, 2H), 7.69 (d, 2H),
7.23 (s, 2H, exchangeable), 6.98 (s, 1H). Anal. (C9H8ClN3O2S2)
C, H, N.
7.22 (s, 2H), 7.03 (s, 1H), 6.89 (d, 1H, 3J ) 1.4 Hz), 3.49 (s, 3H).
Anal. (C13H13N5O2S2‚H2O) C, H, N.
(4-Bromophenyl)[4-(2,4-dimethylimidazol-1-yl)thiazol-2-yl]-
amine (10b). 10b was prepared from (4-chlorothiazol-2-yl)-4-
bromophenylamine (7b) and 2,4-dimethylimidazole as described
above. Yield: 0.22 g (22%), beige crystals. Mp: 213 °C. 1H NMR
(DMSO-d6): δ (ppm) ) 10.59 (s, 1H), 7.57 (d, 2H), 7.51 (d, 2H),
7.13 (s, 1H), 6.87 (s, 1H), 2.42 (s, 3H), 2.08 (s, 3H). Anal. (C14H13-
BrN4S) C, H, N.
(4-Methoxyphenyl)[4-(2,4-dimethylimidazol-1-yl)thiazol-2-yl]-
amine (10c). 10c was prepared from (4-chlorothiazol-2-yl)-4-
methoxyphenylamine (7c) and 2,4-dimethylimidazole as described
above. Yield: 0.22 g (35%), beige crystals. Mp: 155.2 °C. 1H NMR
(DMSO-d6): δ (ppm) ) 10.22 (s, 1H), 7.51 (d, 2H), 7.10 (s, 1H),
6.91 (d, 2H), 6.72 (s, 1H), 3.72 (s, 3H), 2.42 (s, 3H), 2.07 (s, 3H).
Anal. (C15H16N4OS) C, H, N.
Synthesis of Target Derivates 8-12. The appropriate (4-
chlorothiazol-2-yl)arylamine (3.6 mmol) and the imidazole or
benzimidazole derivative (18 mmol) were stirred in 7.5 mL of DMF
at 80 °C for 2-8 days (TLC control). The mixture was cooled to
room temperature and poured into 300 mL of 5% aqueous Na2-
CO3. The crude product was filtered off, dried, and purified by
column chromatography (SiO2, ethyl acetate) if necessary.
(4-Imidazol-1-ylthiazol-2-yl)phenylamine (8a). 8a was prepared
from (4-chlorothiazol-2-yl)phenylamine (7a) and imidazole as
described above. Yield: 2.77 g (80%), beige crystals. Mp: 192
°C. 1H NMR (DMSO-d6): δ (ppm) ) 10.51 (s, 1H), 8.28 (s, 1H),
7.75 (s, 1H), 7.64 (d, 2H), 7.36 (t, 2H), 7.08 (s, 1H), 6.97 (m, 2H).
Anal. (C12H10N4S) C, H, N.
(4-Methylphenyl)[4-(2,4-dimethylimidazol-1-yl)thiazol-2-yl]-
amine (10d). 10d was prepared from (4-chlorothiazol-2-yl)-4-
dimethylphenylamine (7d) and 2,4-dimethylimidazole as described
above. Yield: 0.10 g (22%), beige crystals. Mp: 226 °C. 1H NMR
(DMSO-d6): δ (ppm) ) 10.31 (s, 1H), 7.48 (d, 2H), 7.13 (d, 2H),
7.10 (s, 1H), 6.76 (s, 1H), 2.42 (s, 3H), 2.25 (s, 3H), 2.08 (s, 3H).
Anal. (C15H16N4S) C, H, N.
(4-Imidazol-1-ylthiazol-2-yl)(4-methoxyphenyl)amine (8c). 8c
was prepared from (4-chlorothiazol-2-yl)-4-methoxyphenylamine
(7c) and imidazole as described above. Yield: 0.13 g (63%),
colorless crystals. Mp: 196 °C. 1H NMR (DMSO-d6): δ (ppm) )
10.29 (s, 1H), 8.24 (s, 1H), 7.72 (s, 1H), 7.54 (d, 2H), 7.06 (s,
1H), 6.94 (d, 2H), 6.88 (s, 1H), 3.74 (s, 3H). Anal. (C13H12N4OS)
C, H, N.
(3-Bromophenyl)[4-(2,4-dimethylimidazol-1-yl)thiazol-2-yl]-
amine (10e). 10e was prepared from (4-chlorothiazol-2-yl)-3-
bromophenylamine (7e) and 2,4-dimethylimidazole as described
above. Yield: 0.28 g (57%), beige crystals. Mp: 210-213 °C. 1H
NMR (DMSO-d6): δ (ppm) ) 10.65 (s, 1H), 8.03 (s, 1H), 7.47
(d, 1H), 7.27 (t, 1H), 7.16 (d, 1H), 7.13 (s, 1H), 6.89 (s, 1H), 2.46
(s, 3H), 2.09 (s, 3H). Anal. (C24H13BrN4S) C, H, N.
[4-(2-Methylimidazol-1-yl)thiazol-2-yl]phenylamine (9a). 9a
was prepared from (4-chlorothiazol-2-yl)-4-phenylamine (7a) and
2-methylimidazole as described above. Yield: 0.54 g (58%), beige
crystals. Mp: 197 °C. 1H NMR (DMSO-d6): δ (ppm) ) 10.49 (s,
1H), 7.61 (d, 2H), 7.46 (s, 1H), 7.34 (t, 2H), 6.98 (t, 1H), 6.92 (s,
2H), 2.49 (s, 3H). Anal. (C13H12N4S) C, H, N.
(4-Bromophenyl)[4-(2-methylimidazol-1-yl)thiazol-2-yl]-
amine (9b). 9b was prepared from (4-chlorothiazol-2-yl)04-
bromophenylamine (7b) and 2-methylimidazole as described above.
Yield: 0.33 g (60%), beige crystals. Mp: 271 °C. 1H NMR
(DMSO-d6): δ (ppm) ) 10.62 (s, 1H), 7.59 (d, 2H), 7.49 (d, 2H),
7.43 (d, 1H), 6.95 (s, 1H), 6.88 (d, 1H) 2.47 (s, 3H). Anal. (C13H11-
BrN4S) C, H, N.
(4-Methoxyphenyl)-[4-(2-methylimidazol-1-yl)thiazol-2-yl]-
amine (9c). 9c was prepared from (4-chlorothiazol-2-yl)-4-meth-
oxyphenylamine (7c) and 2-methylimidazole as described above.
Yield: 0.39 g (66%), brown crystals. Mp: 177 °C. 1H NMR
(DMSO-d6): δ (ppm) ) 10.27 (s, 1H), 7.52 (d, 2H), 7.41 (s, 1H),
6.92 (d, 2H), 6.87 (s, 1H), 6.81 (s, 1H), 3.73 (s, 3H), 2.47 (s, 3H).
Anal. (C14H14N4OS) C, H, N.
(4-Methylphenyl)-[4-(2-methylimidazol-1-yl)thiazol-2-yl]-
amine (9d). 9d was prepared from (4-chlorothiazol-2-yl)-4-
methylphenylamine (7d) and 2-methylimidazole as described above.
Yield: 0.20 g (32%), beige crystals. Mp: 219 °C. 1H NMR
(DMSO-d6): δ (ppm) ) 10.37 (s, 1H), 7.48 (d, 2H), 7.41 (s, 1H),
7.13 (d, 2H), 6.87 (s, 1H), 6.85 (s, 1H), 2.47 (s, 3H), 2.25 (s, 3H).
Anal. (C14H14N4S) C, H, N.
(3-Bromophenyl)[4-(2-methylimidazol-1-yl)thiazol-2-yl]-
amine (9e). 9e was prepared from (4-chlorothiazol-2-yl)-3-bro-
mophenylamine (7e) and 2-methylimidazole as described above.
Yield: 0.21 g (43%), beige crystals. Mp: 223 °C. 1H NMR
(DMSO-d6): δ (ppm) ) 10.68 (s, 1H), 8.03 (s, 1H), 7.47 (d, 1H),
7.46 (s 1H), 7.29 (t, 1H), 7.16 (d, 1H), 6.98 (s, 1H), 6.89 (s, 1H),
2.50 (s, 3H). Anal. (C13H11BrN4S) C, H, N.
(3-Methoxyphenyl)[4-(2-methylimidazol-1-yl)thiazol-2-yl]-
amine (9f). 9f was prepared from (4-chlorothiazol-2-yl)-3-meth-
oxyphenylamine (7f) and 2-methylimidazole as described above.
Yield: 0.28 g (48%), brown crystals. Mp: 179 °C. 1H NMR
(DMSO-d6): δ (ppm) ) 10.47 (s, 1H), 7.44 (s, 1H), 7.33 (s, 1H),
7.23 (t, 1H), 7.08 (d, 1H), 6.92 (s, 1H), 6.90 (d, 1H), 6.57 (d, 1H),
3.74 (s, 3H), 2.94 (s, 3H). Anal. (C14H14N4OS) C, H, N.
4-[4-(2-Methylimidazol-1-yl)thiazol-2-ylamino]benzenesulfona-
mide Monohydrate (9g). 9g was prepared from 4-(4-chlorothiazol-
2-ylamino)benzenesulfonamide (7g) and 2-methylimidazole as
described above. Yield: 0.13 g (48%), colorless crystals. Mp:
223.9-225.5 °C. 1H NMR (DMSO-d6): δ (ppm) ) 10.87 (s, 1H),
7.79 (d, 2H), 7.75 (d, 2H), 7.47 (d, 1H), 7.08 (d, 1H, 3J ) 1.4 Hz),
(3-Methoxyphenyl)[4-(2,4-dimethylimidazol-1-yl)thiazol-2-yl]-
amine (10f). 10f was prepared from (4-chlorothiazol-2-yl)-3-
methoxyphenylamine (7f) and 2,4-dimethylimidazole as described
above. Yield: 0.18 g (35%), brown crystals. Mp: 172 °C. 1H NMR
(DMSO-d6): δ (ppm) ) 10.43 (s, 1H), 7.31 (s, 1H), 7.23 (t, 1H),
7.11 (s, 1H), 7.10 (d, 1H), 6.82 (s, 1H), 6.57 (d, 1H), 3.74 (s, 3H),
2.44 (s, 3H), 2.08 (s, 3H). Anal. (C15H16N4OS) C, H, N.
4-(Benzimidazol-1-yl)thiazol-2-yl)phenylamine (11a). 11a was
prepared from (4-chlorothiazol-2-yl)phenylamine (7a) and benz-
imidazole as described above. Yield: 1.7 g (82%), beige crystals.
1
Mp: 229 °C. H NMR (DMSO-d6): δ (ppm) ) 10.58 (s, 1H),
8.76 (s, 1H), 8.00 (d, 1H), 7.78 (d, 1H), 7.67 (d, 2H), 7.37 (m,
4H), 7.17 (s, 1H), 7.02 (t, 1H). Anal. (C16H12N4S) C, H, N.
(4-Bromophenyl)[4-(benzimidazol-1-yl)thiazol-2-yl]amine (11b).
11b was prepared from (4-chlorothiazol-2-yl)-4-bromophenylamine
(7b) and benzimidazole as described above. Yield: 0.31 g (42%),
1
beige crystals. Mp: 272 °C. H NMR (DMSO-d6): δ (ppm) )
10.72 (s, 1H), 8.77 (s, 1H), 7.97 (d, 1H), 7.78 (d, 1H), 7.67 (d,
2H), 7.53 (d, 2H), 7.35 (m, 2H), 7.21 (s, 1H). Anal. (C16H11BrN4S)
C, H, N.
(4-Methoxyphenyl)[4-(benzimidazol-1-yl)thiazol-2-yl]amine
(11c). 11c was prepared from (4-chlorothiazol-2-yl)-4-methoxy-
phenylamine (7c) and benzimidazole as described above. Yield:
0.22 g (33%), brown crystals. Mp: 214 °C. 1H NMR (DMSO-d6):
δ (ppm) ) 10.38 (s, 1H), 8.74 (s, 1H), 7.99 (d, 1H), 7.78 (d, 1H),
7.58 (d, 2H), 7.30-7.43 (m, 2H), 7.08 (s, 1H), 6.97 (d, 2H), 3.75
(s, 3H). Anal. (C17H14N4OS) C, H, N.
[4-(2-Methylbenzimidazol-1-yl)thiazol-2-yl]phenylamine (12a).
12a was prepared from (4-chlorothiazol-2-yl)phenylamine (7a) and
2-methylbenzimidazole as described above. Yield: 0.28 g (22%),
beige crystals. Mp: 130.6 °C (dec). 1H NMR (DMSO-d6): δ (ppm)
) 10.54 (s, 1H, exchangeable), 7.60 (m, 3H), 7.63-7.59 (m, 3H),
7.48-7.43 (m, 3H), 7.34-7.29 (m, 2H), 7.26-7.20 (m, 2H), 7.18
(s, 1H), 6.99-6.95 (m, 1H), 2.60 (s, 3H). Anal. (C17H14N4S) C, H,
N.
(4-Bromophenyl)[4-(2-methylbenzimidazol-1-yl)thiazol-2-yl]-
amine (12b). 12b was prepared from (4-chlorothiazol-2-yl)-4-
bromophenylamine (7b) and 2-methylbenzimidazole as described
above. Yield: 0.07 g (12%), beige crystals. Mp: 231°C. 1H NMR
(DMSO-d6): δ (ppm) ) 10.70 (s, 1H), 7.60 (m, 3H), 7.47 (m, 3H),
7.24 (m, 3H), 2.59 (s, 3H). Anal. (C17H13BrN4S) C, H, N.

5776 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
Mahboobi et al.
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(4-Methoxyphenyl)[4-(2-methylbenzimidazol-1-yl)thiazol-2-
yl]amine (12c). 12c was prepared from (4-chlorothiazol-2-yl)-4-
methoxyphenylamine (7c) and 2-methylbenzimidazole as described
above. Yield: 0.06 g (6%), brown crystals. Mp: 171 °C. 1H NMR
(DMSO-d6): δ (ppm) ) 10.32 (s, 1H), 7.60 (d, 1H), 7.51 (d, 2H),
7.45 (d, 1H), 7.23 (d, 2H), 7.09 (s, 1H), 6.90 (d, 2H), 3.70 (s, 3H),
2.59 (s 3H). Anal. (C18H16N4OS) C, H, N.
(3-Bromophenyl)[4-(2-methylbenzimidazol-1-yl)thiazol-2-yl]-
amine (12e). 12e was prepared from (4-chlorothiazol-2-yl)-3-
bromophenylamine (7e) and 2-methylbenzimidazole as described
above. Yield: 0.57 g (21%), brown crystals. Mp: 241 °C (dec).
1H NMR (DMSO-d6): δ (ppm) ) 10.74 (s, 1H, exchangeable),
8.03-8.01 (m, 1H), 7.65-7.60 (m, 1H), 7.51-7.46 (m, 2H), 7.30-
7.23 (m, 4H), 7.16-7.13 (m, 1H), 2.62 (s, 3H). Anal. (C17H13-
BrN4S) C, H, N.
(3-Methoxyphenyl)[4-(2-methylbenzimidazol-1-yl)thiazol-2-
yl]amine (12f). 12f was prepared from (4-chlorothiazol-2-yl)-3-
methoxyphenylamine (7f) and 2-methylbenzimidazole as described
above. Yield: 0.05 g (10%), beige crystals. Mp: 106 °C. 1H NMR
(DMSO-d6): δ (ppm) ) 10.54 (s, 1H), 7.62 (d, 1H), 7.49 (d, 1H),
7.36 (t, 1H), 7.26-7.21 (m, 3H), 7.18 (s, 1H), 7.07 (d, 1H), 6.55
(d, 1H), 3.70 (s 3H), 2.61 (s 3H). Anal. (C18H16N4OS) C, H, N.
4-[4-(2-Methylbenzimidazol-1-yl)thiazol-2-ylamino]benzensul-
fonamide Hydrochloride Monohydrate (12g). 12g was prepared
from 4-(4-chlorothiazol-2-ylamino)benzenesulfonamide (7g) and
2-methylbenzimidazole as described above. Yield: 0.60 g (23%),
1
beige crystals. Mp: 271.1-272.2 °C. H NMR (DMSO-d6): δ
(ppm) ) 10.95 (s, 1H, exchangeable), 7.78-7.72 (m, 4H), 7.66-
7.60 (m, 1H), 7.49-7.44 (m, 1H), 7.30 (s, 1H), 7.27-7.22 (m,
2H), 7.21 (s, 2H, exchangeable), 2.61 (s 3H). Anal. (C17H15N5O2S2‚
HCl‚H2O) C, H, N.
Supporting Information Available: Experimental details con-
cerning microtubule polymerization assay, cell viability assay, and
colchicine competition binding assay, IR spectral data, EI spectral
data, and the results of combustion analysis. This material is
available free of charge via the Internet at http://pubs.acs.org.
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