Novel microtubule-interacting phenoxy pyridine and phenyl sulfanyl pyridine analogues for cancer therapy

Article abstract of DOI:10.1021/jm800203e

Current microtubule inhibitory agents used in the clinic to treat cancer have severe side effects, and development of resistance is frequent. We have evaluated the antitumor effect of a novel 30-compound library of phenoxy pyridine and phenyl sulfanyl pyridine derivatives. MTT assays revealed that, of all 30 compounds tested, compounds 2 and 3 showed the largest decrease in proliferation (low μM range) against Panc1 and HS766T human pancreatic cancer cells. Flow cytometry experiments with MCF7 breast cancer cells showed a G2/M arrest comparable to that of colcemid. Immunofluorescence staining demonstrated complete disappearance of intracellular microtubules. Tubulin assembly assays, however, showed a dose-dependent decrease in tubulin assembly with compound 3 that seemed limited to about 50% of the control reaction. With compound 2 treatment, there was only a delay in the onset of assembly, with no effect on the extent of the reaction. Taken together, our results show that these novel microtubule inhibitors have promising anticancer activity and can be potentially used to overcome paclitaxel resistance in the clinical setting.

Full text of DOI:10.1021/jm800203e

J. Med. Chem. 2008, 51, 5953–5957
5953
Novel Microtubule-Interacting Phenoxy Pyridine and Phenyl Sulfanyl Pyridine Analogues for
Cancer Therapy
Ravi Kumar Anchoori,† Madeleine Susanne Quirine Kortenhorst,†,‡ Manuel Hidalgo,§ Taradas Sarkar,| Gurulingappa Hallur,†
Ruoli Bai,| Paul J. Van Diest,‡ Ernest Hamel,| and Saeed R. Khan*,†
DiVision of Chemical Therapeutics, The Sidney Kimmel ComprehensiVe Cancer Center at Johns Hopkins, 1650 Orleans Street, CRB-I, 162 C,
Baltimore, Maryland 21231, Pathology Department, UniVersity Medical Center Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, Netherlands,
Gastrointestinal Cancer Program, The Sidney Kimmel ComprehensiVe Cancer Center at Johns Hopkins, 1650 Orleans Street, CRB-I, 162 A,
Baltimore, Maryland 21231, Toxicology and Pharmacology Branch, DeVelopmental Therapeutics Program, DiVision of Cancer Treatment and
Diagnosis, National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland 21702
ReceiVed February 26, 2008
Current microtubule inhibitory agents used in the clinic to treat cancer have severe side effects, and
development of resistance is frequent. We have evaluated the antitumor effect of a novel 30-compound
library of phenoxy pyridine and phenyl sulfanyl pyridine derivatives. MTT assays revealed that, of all 30
compounds tested, compounds 2 and 3 showed the largest decrease in proliferation (low µM range) against
Panc1 and HS766T human pancreatic cancer cells. Flow cytometry experiments with MCF7 breast cancer
cells showed a G2/M arrest comparable to that of colcemid. Immunofluorescence staining demonstrated
complete disappearance of intracellular microtubules. Tubulin assembly assays, however, showed a dose-
dependent decrease in tubulin assembly with compound 3 that seemed limited to about 50% of the control
reaction. With compound 2 treatment, there was only a delay in the onset of assembly, with no effect on the
extent of the reaction. Taken together, our results show that these novel microtubule inhibitors have promising
anticancer activity and can be potentially used to overcome paclitaxel resistance in the clinical setting.
Introduction
are known to inhibit tubulin polymerization, cause microtubule
depolymerization in vitro, and demonstrate activity against solid
tumors.5,6
Cancer is a major worldwide health problem. Improvements
in treatment and prevention have led to a decrease in cancer
deaths, but the number of new diagnoses continues to rise.
Treatment of cancer cells with agents that interfere with
microtubule assembly causes mitotic arrest and eventually cell
death. Current microtubule inhibitory agents used in the clinic
have severe side effects, and development of resistance is
frequent. We have designed and synthesized a novel 30-
compound library of phenoxy pyridine (PPa) and phenyl sulfanyl
pyridine (PSP) derivatives and studied their effects in pancreatic
cancer, breast cancer, and Burkitt lymphoma cells.
N,N-dimethylamino-benzoylphenylurea 16 is a novel, small-
molecule, orally available, tubulin-interactive agent that is
currently undergoing phase I clinical evaluation in refractory
solid tumors in humans. When administered on a continuous
weekly schedule, the dose-limiting toxicity (DLT) of NSC
639829 was severe myelosuppression. This DLT correlated with
continual accumulation of the parent compound and cytotoxic
metabolites. An alternative approach of an interrupted schedule
(6 weeks on/2 weeks off) appears to prevent severe myelosup-
pression while maintaining antitumor efficacy.
Our strategy, as outlined in this paper, was to discover new
microtubule inhibitors using a small molecule library of
compounds that contained the PP or PSP core structures.
Previously, we published work from our laboratory related to
design, synthesis, and evaluation of novel benzoylphenylurea
(BPU) compounds as microtubule inhibitors.1,2 BPU and its
derivatives were originally developed as insecticides,3,4 but they
were later found to possess cytotoxic activity.4 BPU derivatives
* To whom correspondence should be addressed. Phone: (410)614-0200.
Fax: (410)614-8397. E-mail: khansa@jhmi.edu.
†
Division of Chemical Therapeutics, The Sidney Kimmel Comprehen-
sive Cancer Center at Johns Hopkins.
‡
Pathology Department, University Medical Center Utrecht.
Gastrointestinal Cancer Program, The Sidney Kimmel Comprehensive
§
Cancer Center at Johns Hopkins.
|
Toxicology and Pharmacology Branch, Developmental Therapeutics
Program, Division of Cancer Treatment and Diagnosis, National Cancer
Institute at Frederick, National Institutes of Health.
a Abbreviations: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide; PP, phenoxy pyridine; PSP, phenyl sulfanyl pyridine; BPU,
benzoyl phenyl urea; DLT, dose limiting toxicity; DMF, dimethyl forma-
mide; SOCl2, thionyl chloride; MPM2, mitotic protein monoclonal 2; DAPI,
4,6-diamidino-2-phenylindole.
10.1021/jm800203e CCC: $40.75
2008 American Chemical Society
Published on Web 09/09/2008

5954 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Chart 1. Structure of PP and PSP Analogues
Anchoori et al.
Table 1. Growth Inhibition of Pancreatic Cancer Cell Lines by PP and
PSP Analogues Compared with Untreated Control
Scheme 1
Panc1
HS766T
analogues 1 µM SD 10 µM SD 1 µM SD 10 µM SD
14
15
16
17
18
19
2
20
22
23
24
25
26
27
28
29
3
30
31
32
33
35
36
37
38
39
40
41
42
74
70
58
76
69
66
47
48
86
67
88
61
89
63
62
97
35
77
60
65
98
63
64
54
72
53
80
96
36
11
12
15
24
2
7
7
6
7
11
8
10
7
14
12
6
4
11
7
9
9
58
44
68
55
46
51
38
43
66
53
44
53
51
51
41
68
33
52
38
41
63
60
43
42
66
58
81
88
79
7
5
16
17
6
6
4
7
5
6
3
5
8
8
8
8
4
7
4
2
3
6
5
4
11
4
77
50
107
55
109
76
36
71
95
79
101
67
97
79
105
88
30
54
63
77
106
81
18
10
40
7
34
18
5
16
10
9
19
15
21
15
18
9
6
12
5
19
19
12
8
21
9
66
27
64
30
36
70
23
56
74
73
23
50
75
62
87
73
21
31
23
26
31
79
66
93
83
68
101
97
26
18
5
7
4
6
14
2
16
15
14
2
12
15
10
8
15
2
3
2
3
2
13
7
7
12
6
14
14
1
In the meantime, BPU analogues are being synthesized to
optimize potency and improve physicochemical properties. This
effort has led to the development of a highly potent, novel series
of modified sulfur BPU compounds. These analogues were
synthesized in excellent yield by replacing the urea moiety with
thiourea and the ether moiety with sulfide, sulfoxide, or sulfone
groups by coupling corresponding benzoylisothiocyanate and
aniline derivatives. Preliminary investigation of the most active
of the sulfur analogues demonstrated excellent in vitro and in
vivo efficacy against pancreas, prostate, and breast cancer
models.1 Sulfur analogues of BPU appear to be promising
compounds as a successor to 1. However, development of the
sulfur analogues is limited by their poor solubility in solvents
generally used in formulations. The sulfur analogues are also
difficult to formulate for oral administration.
5
9
86
74
78
80
97
96
87
12
14
5
12
12
3
16
13
14
16
12
9
11
moiety is present. As expected, these compounds show activity
in the low µM range on pancreatic cell lines. The most active
compounds were 2 and 3, and they were selected for further
studies.
Results and Discussion
On the basis of these findings, we concluded that it would
be desirable to change the scaffold of the BPU molecule while
keeping the carbonyl urea moiety intact. A literature search led
us to the molecule sorafenib from the Bayer Company. Sorafenib
is structurally related to the BPUs, but it is a raf-kinase inhibitor
and is currently marketed for the treatment of cancer. We
therefore modified our sulfur BPU analogue by introducing
substituted phenoxy or thiophenyl moieties and synthesized a
library of 30 PP and PSP compounds (Chart 1). We evaluated
these compounds for in vitro activity against pancreatic cancer
cell lines.
Chemistry. Synthesis of the library compounds was based
in Scheme 1. 2-Picolinic acid 4 was treated with SOCl2 in DMF,
yielding acid chloride 5 as the HCl salt, which on treatment
with the indicated amines in methanol yielded compounds of
general structure 6. Further treatment of compound 6 with
4-amino phenol and 4-amino thiophenol of general formula 7
in the presence of potassium tert-butoxide yielded anilines 8 as
the major and 9 as the minor products. Substituted isocyanates
12 were synthesized by treating corresponding amides 11 with
oxalyl chloride in the presence of dichloroethane. The free amine
functionality of general structure 8 treated with corresponding
isocyanates 12 and isothiocyanate (purchased from SIGMA) 13
in the presence of dioxane yielded target compounds 10. The
The compounds differ in substitution pattern and in the
presence of hetero atoms, but in all cases the carbonyl urea

Microtubule-Interacting PP and PSP Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 5955
Figure 1. Treatment of MCF7 cells for 15 h with either 5 µM compound 2 or 5 µM compound 3 results in a 10-15-fold increase in mitotic cells.
Colcemid (0.25 µM) was used as a positive control. In the propidium iodide scans, M1 indicates G1 cells, M3 cells in S phase, and M2 cells at
G2/M.
structures of the library compounds are shown in Chart 1. All
compounds were characterized by spectral data that confirmed
the assigned structures.
fold increase in mitotic cells, comparable to the effect observed
with colcemid.
We next wished to confirm that tubulin was the direct target
of 2 and 3, and we examined the effects of these agents on the
polymerization of purified tubulin. We used a reaction condition8
that has been used successfully with many different antitubulin
agents binding at various sites on the tubulin molecule, both in
its unpolymerized Rꢀ-heterodimer form and in microtubules.
Generally, a concentration that inhibits extent of assembly at
20 min by 50%, as measured by turbidity development, is
determined8 (see Figure 2C for a typical inhibition pattern,
obtained with 1.5-4.0 µM colchicine; a series of such experi-
ments yielded an IC50 of 2.2 µM for colchicine). However, this
parameter could not be measured for 2. As shown in Figure
2A, with selected concentrations of 2, assembly was progres-
sively delayed in its onset with up to about 4 µM 2. In addition,
initially there were also decreasing rates of polymer propagation
with increasing concentrations of 2. At no concentration was a
significant effect observed on extent of assembly. At concentra-
tions over about 4 µM, there was a seemingly paradoxical
reversal of the effects on assembly onset and propagation rates,
although at all concentrations examined, there was some effect
on the overall assembly reaction. This may indicate formation
of a polymer of aberrant morphology, as occurs with a variety
of antitubulin compounds. These unusual effects may also be
Biology. PP and PSP Analogues in the Low µM Range
Decrease Proliferation of Pancreatic Cancer Cells. Table 1
compares the cytotoxicity of the PP and PSP derivatives in an
initial screen at 1 and 10 µM against human pancreatic cancer
cell lines Panc1 and HS766T, as determined with the MTT
assay. Because only 2 and 3 had submicromolar IC50s in both
cell lines, further evaluation was limited to these two agents.
These studies showed that 2 had IC50 values of 0.8 and 0.15
µM against Panc1 and HS766T cells, respectively, and that 3
had IC50 values of 0.7 and 0.5 µM against Panc1 and HS766T
cells, respectively.
PP and PSP Analogues Cause G2/M Arrest. Because the
compounds were designed to bind to tubulin, flow cytometry
experiments7 were carried out to determine whether compound
treatment would lead to cell cycle arrest at G2/M. MCF7 breast
cancer cells were treated for 15 h with 5 µM 2 or 5 µM 3. As
a positive control, 0.1 µg/mL (about 0.25 µM) colcemid was
used. After incubation, cells were harvested and fixed prior to
labeling of DNA with propidium iodide and labeling of mitotic
cells with the monoclonal antibody MPM2. MPM2 specifically
recognizes the phosphorylated epitopes of proteins that are
phosphorylated at the onset of mitosis. Figure 1 shows that
treatment of MCF7 cells for 15 h with 2 or 3 caused 10-13

5956 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Anchoori et al.
There was a slight suggestion in some experiments, as shown
in Figure 2B, of a similar paradoxical rise in the turbidity
readings at the highest concentrations of 3. The greater, although
still limited, effect of 3 on tubulin assembly may indicate greater
solubility of this compound as compared with 2 in 0.8 M
glutamate.
The difficulty of quantitating inhibition of the assembly
reaction based on its extent, especially with 2, led us to
determine instead compound concentrations that inhibited the
maximum propagation rate by 50%. The turbidity data were
evaluated with Microcal Origin, which was used to calculate
the first derivative for each reading (4 readings per sample
per minute). The maximum value was determined for the
controls and for each compound concentration. We obtained
the following results, expressed in terms of IC50 ( standard
deviation, versus control reaction: for colchicine, 0.99 ( 0.01
µM; for 2, 4.0 ( 0.1 µM; for 3, 1.7 ( 0.3 µM.
As shown in the figure, we did not find any cells in
metaphase, anaphase or telophase. Figure 3B therefore shows
only a control cell in metaphase. Not all cells were in mitosis
after the 15 h treatment. Some interphase cells displayed
several micronuclei, indicating escape from the mitotic
spindle checkpoint. Colcemid at 0.25 µM was again used as
a positive control.
Immunofluorescence studies were done because of the
seeming contradiction between robust G2/M arrest and the
equivocal tubulin assembly data of Figure 2.
Figure 2. Effects of varying concentrations of 2 (A), 3 (B), and
colchicine (C) on tubulin assembly. At zero time, the temperature was
rapidly increased from 0 to 30 °C. Compound concentrations were as
follows: (A) curve 1, control (no compound); curves 2-6, 2 at 1.5,
4.0, 10, 20, and 40 µM, respectively; (B) curve 1, control (no
compound); curves 2-6, 3 at 1.5, 4.0, 10, 20, and 40 µM, respectively;
(C) curve 1, control (no compound); curves 2-6, colchicine at 1.0,
1.5, 2.0, 3.0, and 4.0 µM, respectively.
PP and PSP Analogues Cause Complete Disassembly of
Cellular Microtubule. To obtain further information about the
mitotic arrest caused in MCF7 cells by 2 and 3, we performed
immunofluorescence experiments7 with an antibody against
R-tubulin, with cellular DNA stained with 4,6-diamidino-2-
phenylindole (DAPI). Concordant with our flow cytometry
experiments, a large number of cells were arrested in mitosis
after 15 h treatment with either 5 µM 2 or 5 µM 3. All mitotic
cells were arrested in prometaphase of the cell cycle (Figure
3A).
caused by limited solubility of 2 in the 0.8 M glutamate required
to induce assembly.
A somewhat different pattern was observed with 3 (Figure
2B) in that the turbidity readings with 4-5 µM compound were
below 50% of the control value, but all higher concentrations
of 3 reached an apparent limit of about 40% of the control value.
Effect of 3 Compared to Paclitaxel on Growth of
SKOV3 and SKOV3-Paclitaxel-Resistant Cells. We further
studied dose response of SKOV3 and SKOV3-paclitaxel-
Figure 3. Immunofluorescence staining of MCF7 cells treated for 15 h with either 5 µM 2, 5 µM 3, or 0.25 µM colcemid. Tubulin was labeled
with primary antibody against R-tubulin from Molecular Probes and secondary antibody Alexa-568. DNA was stained with DAPI. (A) Loss of
cellular microtubules and prometaphase arrest in MCF7 cells treated for 15 h. (B) Metaphase plate formation in untreated control cells. (C) Micronuclei
formation in treated interphase cells that bypassed the mitotic checkpoint.

Microtubule-Interacting PP and PSP Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 5957
Figure 4. Cytotoxicity assay of 3 and paclitaxel on SKOV3 and SKOV3-paclitaxel-resistant ovarian cell lines.
resistant cells to 3 in comparison with paclitaxel for 7 days
(Figure 4). Both 3 and paclitaxel were evaluated by using
standardcytotoxicityassaysinSKOV3andSKOV3-paclitaxel-
resistant ovarian cancer cell lines. Even though 3 is less
effective in SKOV3 cell lines, it showed excellent cytotox-
icity compared to that of the paclitaxel at equivalent doses
in paclitaxel-resistant cell lines. Further mechanistic studies
are ongoing.
with promising anticancer activity that retains the ability to
induce cell death in a paclitaxel-resistant cell line.
Acknowledgment. We gratefully acknowledge the financial
support of grants from FAMRI and NIH. We thank the
Medicinal Chemistry Center of the SKCCC at Johns Hopkins
for conducting the NMR and LC-MS studies. In addition, we
thank Susan Dalrymple and Dr. Antonio Jimeno, Sidney Kimmel
Comprehensive Cancer Center at Johns Hopkins, for help in
cell culture analysis.
Conclusion
On the basis of our antiproliferative study of the library
compounds, it appears that the substituent pattern on the “left-
hand” (see structural diagram) phenyl ring is critical for activity,
with the chlorine atom at position 2 being particularly important,
but not sufficient, for activity. This -Cl group was present in
both of the most active compounds, and it was the sole
substituent on the phenyl ring in 2, but this same -Cl substituent
was also present in many of the less active agents. It should
also be noted that the amide substituent differed in 2 and 3,
and the former was an ether (“X” group in the diagram) and
the latter a thioether. 18, the cognate thio ether of 2, was less
active than 2, but the cognate ether of 3 has not yet been
prepared.
In conclusion, we have synthesized a series of PP and PSP
analogues. Some of these had excellent growth inhibition activity
against both pancreatic and breast cancer cell lines, and 3 and
2 were the most potent. Cell cycle analysis on breast cancer
MCF7 cells treated with 2 or 3 revealed that both agents caused
mitotic arrest. These compounds are currently being evaluated
for their in vivo efficacy in animal models. These findings have
encouraged us to continue the development and testing of novel
analogues and to conduct further studies to investigate SAR
and their mechanisms of action.
Supporting Information Available: Spectral data for all the
compounds and experimental procedures for biological evaluation.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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Further, 3 showed significant cytotoxicty against a paclitaxel-
resistant cell lines as compared with paclitaxel at equivalent
doses. Our results show that these analogues inhibited cell
survival in both the paclitaxel-sensitive and paclitaxel-resistant
ovarian cancer cells (SKOV3) with equivalent activity. Further
mechanistic evaluation of the most potent analogues is under-
way. In summary, we have identified a small molecule inhibitor
JM800203E