2-Aryl-4,5,6,7-tetrahydro-1,3-benzothiazol-7-ols as a class of antitumor agents selectively active in securin-/- cells

Article abstract of DOI:10.1016/j.bmcl.2010.05.021

A series of 2-(4-aminophenyl)-4,5,6,7-tetrahydro-1,3-benzothiazol-7-ols have been developed as antitumor agents that showed high selectivity against aneuploid cell lines (vs diploid cell lines). Structure-activity relationship studies showed that a hydroxymethyl group at the 2-position of the phenyl ring increased potency and selectivity. A pyrrolidinyl group at the 4-position of the phenyl ring was comparable to a dimethylamino group. The corresponding 5-aza analogs, 2-(4-aminophenyl)-4,5,6,7-tetrahydro[1,3]thiazolo[4,5-c]pyridin-7-ols, retained potency and high level of selectivity against aneuploid cell growth (vs diploid cells). These 5-aza compounds exhibited higher water solubility and higher metabolic stability than the corresponding carba analogs. Compound 19 showed the highest potency against MCF-7 and MDA-MB-361 lines and was selected for further evaluation.

Full text of DOI:10.1016/j.bmcl.2010.05.021

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3903–3905
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Aryl-4,5,6,7-tetrahydro-1,3-benzothiazol-7-ols as a class of antitumor
agents selectively active in securinÀ/À cells
a,
a
a
b
Nan Zhang a, , Semiramis Ayral-Kaloustian , Chuansheng Niu , Thai Nguyen , Erik Upeslacis ,
*
*
Tarek S. Mansour a, Shoba Ragunathan b, Edward Rosfjord b
a Discovery Medicinal Chemistry, Chemical Sciences, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA
b Oncology Research, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 January 2010
Revised 7 May 2010
Accepted 10 May 2010
Available online 12 May 2010
A series of 2-(4-aminophenyl)-4,5,6,7-tetrahydro-1,3-benzothiazol-7-ols have been developed as antitu-
mor agents that showed high selectivity against aneuploid cell lines (vs diploid cell lines). Structure–
activity relationship studies showed that a hydroxymethyl group at the 2-position of the phenyl ring
increased potency and selectivity. A pyrrolidinyl group at the 4-position of the phenyl ring was compa-
rable to a dimethylamino group. The corresponding 5-aza analogs, 2-(4-aminophenyl)-4,5,6,7-tetrahy-
dro[1,3]thiazolo[4,5-c]pyridin-7-ols, retained potency and high level of selectivity against aneuploid
cell growth (vs diploid cells). These 5-aza compounds exhibited higher water solubility and higher met-
abolic stability than the corresponding carba analogs. Compound 19 showed the highest potency against
MCF-7 and MDA-MB-361 lines and was selected for further evaluation.
Keywords:
Securin knockout
Aneuploidy
Ó 2010 Elsevier Ltd. All rights reserved.
Chromosomal instability is a pathological process common to
most solid tumors and results in chromosomal rearrangement
and aneuploidy. The mitotic spindle checkpoint is the major cell-
cycle regulatory mechanism controlling the transition from meta-
phase to anaphase and functions to maintain euploidy (diploidy).
As a consequence, loss or mutation of the protein components of
the mitotic spindle checkpoint, such as Mad2, Bub1, BubR1, and
securin, result in chromosomal instability.1–4 Securin regulates a
proteolytic cleavage cascade that permits chromosome segregation
in anaphase.5,6 Loss of securin protein by genetic knockout results
in impaired activation of the proenzyme form of separase and inhi-
bition of this proteolytic cascade. This impairs sister chromatid
separation in securinÀ/À cells, ultimately leading to the malsegre-
gation of sister chromatids and aneuploidy.4 Chromosomal insta-
bility allows for the amplification of oncogenes and the silencing
of growth suppression genes (e.g., p53), resulting in uncontrolled
cell growth.
This unregulated growth makes cancer cells more sensitive to
DNA damaging agents, topoisomerase inhibitors, and microtubule
polymerization inhibitors.7 Recent evidence suggests that reliance
on the unfolded protein response and dependence on protein fold-
ing chaperones makes aneuploid cells more sensitive to these
inhibitors as well.7 Since chromosomal instability is a complex cel-
lular process and the cellular consequences are unclear, we used a
cell-based screening system to identify compounds that selectively
inhibited the growth of chromosomally unstable cells while spar-
ing diploid cells. The isogenic securinÀ/À cells (clones D8 and F3)
generated from the diploid HCT116 colon cancer cell line4 demon-
strate chromosomal instability with >80% of securinÀ/À cells exhib-
iting a loss of at least one chromosome. In this Letter, we report the
discovery and the SAR of a novel series of compounds that selec-
tively inhibit the growth of securinÀ/À cells and other aneuploid
cell types, while permitting the growth of securin wild type cells
and other diploid cell types.
The synthesis of 2-[4-(dimethylamino)phenyl]-4,5,6,7-tetrahy-
dro-1,3-benzothiazol-7-ol (3) is shown in Scheme 1. Starting from
4-(dimethylamino)benzonitrile (1), treatment with P2S5 led to
4-(dimethylamino)thiobenzamide (2). Cyclization with 7-oxabicy-
S
a
N
N
NC
H2N
1
2
OH
b
S
N
N
3
* Corresponding authors. Tel.: +1 845 602 3425; fax: +1 845 602 5561.
E-mail addresses: zhangn@wyeth.com (N. Zhang), ayralks@verizon.net (S. Ayral-
Kaloustian).
Scheme 1. Synthesis of 2-[4-(dimethylamino)phenyl]-4,5,6,7-tetrahydro-1,3-ben-
zothiazol-7-ol (3). Reagents and conditions: (a) P2S5 (0.5 equiv), MeOH, rt, 18 h,
70%; (b) 7-oxabicyclo[4.1.0]heptan-2-one (2 equiv), MeOH, 40 °C, 18 h, 45%.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.021

3904
N. Zhang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3903–3905
OH
OH
OMs
N
clo[4.1.0]heptan-2-one gave the desired compound 3 in racemic
form. Chiral HPLC separation provided the (R)- and (S)-enantio-
mers of 3 (3a and 3b, respectively). Analogs with R1, R2 substitu-
ents on the phenyl ring (Table 1) were prepared in a similar
manner from the corresponding substituted benzonitriles.8
a
b
c
N
t- Boc
7
N
H
N
t-Boc
5
t-Boc
As shown in Scheme 2, synthesis of 2-[4-(dimethyl-
amino)phenyl]-4,5,6,7-tetrahydro[1,3]thiazolo[4,5-c]pyridin-7-ol
(12) started from 4-hydroxypiperidine (4). Protection of the amino
group with t-Boc followed by mesylation and subsequent elimina-
tion gave 3,6-dihydro-1(2H)-pyridine (7). Epoxidation followed by
ring-opening and elimination led to 3-hydroxy-3,6-dihydro-1(2H)-
pyridine (9).9 Epoxidation of 9, followedby oxidation of the hydroxyl
group in intermediate 10, provided 5-oxo-7-oxa-3-azabicy-
clo[4.1.0]heptane (11), which was coupled with 4-(dimethyl-
amino)thiobenzamide (2). Removal of the t-Boc group gave the
desired final compound 2-[4-(dimethylamino)phenyl]-4,5,6,7-tet-
rahydro[1,3]thiazolo[4,5-c]pyridin-7-ol (12). Analogs with R1, R2
substituents on the phenyl ring (Table 2) were prepared in a similar
manner from the corresponding substituted benzonitriles.10
As shown in Table 1, (R)-2-[4-(dimethylamino)phenyl]-4,5,6,7-
tetrahydro-1,3-benzothiazol-7-ol (3a) exhibited high selectivity
in inhibiting aneuploid (vs diploid) cell growth.11,12 Its (S)-enantio-
mer (3b), however, was much less potent in the aneuploid cell
lines, suggesting a very specific protein binding site, with prefer-
ence for the (R)-configuration of the hydroxyl group. Further bio-
logical studies are needed to identify this site in cells. Addition of
a methyl group at the 2-position on the phenyl ring increased po-
tency in aneuploid cell lines (13 vs 3 and 13a vs 3a), while a
hydroxylmethyl group at the same position further increased po-
tency in aneuploid cell lines (15 vs 13 and 3; 16 vs 14). A pyrrolid-
inyl group at the 4-position on the phenyl ring seemed to afford
higher potency in aneuploid cell lines (16 vs 15).
While 2-(4-aminophenyl)-4,5,6,7-tetrahydro-1,3-benzothiazol-
7-ols showed high potency (against aneuploid cell lines) and selec-
tivity (aneuploid cell lines vs. diploid cell lines), they tended to
have low water-solubility and metabolic stability (see below).
The 5-aza analogs (12, 17–19) were prepared to increase polarity
in hope of improving water-solubility and possibly metabolic sta-
bility. To our delight, these 5-aza analogs were able to retain the
potency (against aneuploid cell lines) and selectivity (vs diploid
cell lines). Compound 19 proved to be the most potent against
MCF-7 and MDA-MB-361 lines, and highly selective.11,12 As we ex-
pected, these 5-aza compounds exhibited much higher solubility in
4
6
O
O
OH
OH
d
f
e
N
N
N
t-Boc
t-Boc
9
t-Boc
10
8
OH
O
O
g
h
i
S
N
HN
N
N
t-Boc
12
11
Scheme 2. Synthesis of 2-[4-(dimethylamino)phenyl]-4,5,6,7-tetrahydro[1,3]thiaz-
olo[4,5-c]pyridin-7-ol (12). Reagents and conditions: (a) t-Boc2O; (b) CH3SO2Cl; (c)
DBU; (d) mCPBA; (e) TMSI; then DBU; (f) mCPBA (2 equiv), CH2Cl2, rt, 12 h, 94%; (g)
Dess–Martin (1.2 equiv), CH2Cl2, rt, 12 h, 77%; (h) 2 (1.0 equiv), EtOH, 60 °C, 16 h,
45%; (i) 4 N HCl/dioxane, rt, 12 h, 79%.
carba analogs. Oxidation at the 5- nitrogen was the major meta-
bolic pathway for the 5-aza analogs, while dealkylation of the ani-
line group appeared to be the major metabolic pathway for the
carba analogs. In nude mouse microsomes, the t1/2 for 12, 17–19
ranged from 13 to 26 min, while it was 3 min or less for 3, 13–
16. Pharmacokinetic studies in mice showed that, when dosed or-
ally as a free base at 50 mg/kg, compound 19 had a plasma level of
232 ng/mL at 1 h and 9 ng/mL at 4 h, and an oral bioavailability of
18% (36% when dosed as a HCl salt).
In conclusion, we have developed 2-(4-aminophenyl)-4,5,6,7-
tetrahydro-1,3-benzothiazol-7-ol and their 5-aza analogs, 2-(4-
aminophenyl)-4,5,6,7-tetrahydro[1,3]thiazolo[4,5-c]pyridin-7-ols,
that demonstrated high levels of selectivity against aneuploid cell
growth (vs diploid cells). Introduction of the 5-aza moiety provided
higher water solubility and higher metabolic stability, compared
with the corresponding carba analogs. The aza-analog 19 showed
the highest potency against MCF-7 and MDA-MB-361 cell lines.
Owing to this high potency, accompanied by the very high selectiv-
ity observed against the securinÀ/À cells as well as the breast can-
cer cells, compound 19 was chosen for further biological
mechanism of action studies and in vivo evaluations.
water at pH 7.4 (20 to >100
lg/mL for 12, 17–19) than their carba
analogs (2 to 15 g/mL for 3, 13–16). More importantly, the 5-aza
l
analogs showed much higher metabolic stability relative to their
Table 1
Inhibition of cell growth by 2-(4-aminophenyl)-4,5,6,7-tetrahydro-1,3-benzothiazol-7-ols 3, 13–16
R1
OH
S
*
NR2
N
R1
NR2
IC50 (lM)
a
Compd
*
HCT 116
D8
1.5
0.85
20
0.56
0.27
1.53
0.06
F3
3.0
1.43
46
1.16
0.23
2.80
0.06
0.044
MCF-10A
MCF-7
MDA-MB-361
3
Racemic
R
S
Racemic
R
Racemic
Racemic
Racemic
H
H
H
NMe2
NMe2
NMe2
NMe2
NMe2
1-Pyrrolidinyl
NMe2
>150
114
>100
127
>100
>100
78
>150
>100
>100
>100
>100
>100
>100
17
1.17
1.45
3.30
2.15
0.73
8.70
0.36
0.056
0.83
0.20
3a
3b
13
13a
14
15
16
CH3
CH3
H
CH2OH
CH2OH
0.16
0.083
0.20
0.42
0.032
1-Pyrrolidinyl
21
0.042
a
Determinations were made at 10 concentrations, in triplicate (except for in duplicate), and repeat values agreed, on average, within 40%.

N. Zhang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3903–3905
3905
Table 2
Inhibition of cell growth by 2-[4-(dimethylamino)phenyl]-4,5,6,7-tetrahydro[1,3]thiazolo[4,5-c]pyridin-7-ols 12, 17–19
R1
OH
S
NR2
HN
N
R1
NR2
IC50
(lM)
a
Compd
HCT 116
D8
F3
MCF-10A
MCF-7
MDA-MB-361
12
17
18
19
H
H
NMe2
1-Pyrrolidinyl
NMe2
121
25
>100
>100
1.33
0.73
0.10
0.07
1.63
1.07
0.1
70.2
27.8
70
1.60
1.53
0.031
0.013
0.17
0.14
0.022
0.020
CH2OH
CH2OH
1-Pyrrolidinyl
0.06
>100
a
Determinations were made at 10 concentrations, in triplicate, and repeat values agreed, on average, within 40%.
5. Nasmyth, K. Annu. Rev. Genet. 2001, 35, 673.
6. Pines, J. Trends Cell Biol. 2006, 16, 55.
Acknowledgments
7. Williams, B. R.; Amon, A. Cancer Res. 2009, 69, 5289.
8. Further synthetic details on this series can be found in Zhang, N., et al. US
Patent Application 2009/02,70,363.
9. Compound 9 can also be obtained from commercial sources.
10. Further synthetic details on this series can be found in Zhang, N., et al. US
Patent Application 2009/02,70,447.
The authors thank Dr. Philip Frost for his support of this project,
Mr. Murthy Damarla for chiral HPLC separations, Drs. Joseph Ash-
croft and Thomas Williamson for structural elucidations of the
enantiomers through NMR and CD studies, respectively, and Dr. In-
der Chaudhary for the PK and DSM studies.
11. Cell culture: Cells were maintained in RPMI 1640 medium containing 10% fetal
bovine serum and 50 lg/ml gentamicin at 37 °C in a humidified incubator,
under 5% CO2. HCT116 securin+/+ and the securinÀ/À cell lines D8 and F3 were
obtained from Prof. Bert Vogelstein (Johns Hopkins University, Baltimore, MD).
Aneuploid breast cancer cell lines MDA-MB-361 and MCF-7 and the diploid
breast epithelial cell line MCF-10A were obtained from the American Type
Culture Collection.
References and notes
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J. M. J. Cell Biol. 2003, 160, 341.
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A.; Kumar, R.; Jenkins, R. B.; de Groen, P. C.; Roche, P.; van Deursen, J. M. Nat.
Genet. 2004, 36, 744.
3. Michel, L. S.; Liberal, V.; Chatterjee, A.; Kirchwegger, R.; Pasche, B.; Gerald, W.;
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12. Cell Proliferation assays: Cells were plated in 96-well tissue culture plates. The
next day, dilutions of compound (final concentrations 2 nM to 200 lM) were
added and cells were cultured for 2 days. Cell survival was determined using
the methods described in Rabindran, S. K.; Discafani, C. M.; Rosfjord, E. C.;
Baxter, M.; Floyd, M. B.; Golas, J.; Hallett, W. A.; Johnson, B. D.; Nilakantan, R.;
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