Tricyclic thiazoles are a new class of angiogenesis inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.02.067

Tricyclic thiazoleamine derivatives that were identified as hits in a screen against human umbilical vein endothelial cell proliferation were subjected to a structure-activity relationship study. Two structurally superimposable scaffolds - 4H-thiochromeno[4,3-d]thiazol-2-amine and 5,6-dihydro-4H-benzo[6,7]cyclohepta[1,2-d]thiazol-2-amine derivatives - yielded low-micromolar inhibitors, and two among them 37 and 43 also exhibited antiangiogenic activity in an endothelial tube formation assay. Thus, 37 and 43 can serve as leads to develop a novel class of antiangiogenic agents.

Full text of DOI:10.1016/j.bmcl.2013.02.067

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2733–2737
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Tricyclic thiazoles are a new class of angiogenesis inhibitors
Shridhar Bhat a, Joong Sup Shim a, Jun O. Liu a,b,
⇑
a Department of Pharmacology & Molecular Sciences, 725 North Wolfe Street, Baltimore, MD 21205, USA
b Department of Oncology, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Tricyclic thiazoleamine derivatives that were identified as hits in a screen against human umbilical vein
endothelial cell proliferation were subjected to a structure–activity relationship study. Two structurally
superimposable scaffolds—4H-thiochromeno[4,3-d]thiazol-2-amine and 5,6-dihydro-4H-benzo[6,7]cyclo
hepta[1,2-d]thiazol-2-amine derivatives—yielded low-micromolar inhibitors, and two among them 37
and 43 also exhibited antiangiogenic activity in an endothelial tube formation assay. Thus, 37 and 43
can serve as leads to develop a novel class of antiangiogenic agents.
Received 4 January 2013
Revised 6 February 2013
Accepted 13 February 2013
Available online 24 February 2013
Keywords:
Ó 2013 Elsevier Ltd. All rights reserved.
Tricyclic thiazoles
Endothelial cells
Angiogenesis
Angiogenesis plays an essential role in tumor growth and metas-
tasis. Inhibitors of angiogenesis are emerging as a new class of anti-
cancer drugs.1–3 In the clinic, it has been found that inclusion of an
antiangiogenic drug like bevacizumab, sunitinib, or sorafenib in the
combination chemotherapy produces significant survival bene-
fits4,5 and hence, antiangiogenic drugs have become an integral part
of front-line therapy in treating different types of cancers. Unfortu-
nately, primary and acquired resistance to antiangiogenic therapy is
becoming a real impediment and new agents with novel mecha-
nisms of action are urgently needed to tackle this problem.6 Because
proliferation of endothelial cells is an obligatory step for in vivo
angiogenesis, a direct growth inhibition of endothelial cells in cul-
ture has come to serve as a proxy for anti-angiogenesis screening.
During a routine test of target compounds and intermediates
synthesized in our laboratory, we discovered that four tricyclic thi-
azoles—3, 7, 9, and 11 (Table 1)—were moderately antiproliferative
against human umbilical vein endothelial cells (HUVEC) in a [3H]-
thymidine incorporation assay. In fact, these tricyclic thiazoles
had been synthesized in the course of our development of methio-
nine aminopeptidase (MetAP) inhibitors as antimycobacterial
agents.7 It has been established earlier using fumagillin that inhibi-
tion of human MetAP2 leads to the growth inhibition of HUVEC.8,9
However, tricyclic thiazoles 3, 7, 9, and 11 did not inhibit (up to
typical examples are shown in Scheme 1. Briefly, condensation of
6-chlorothiochroman-4-one (Eq. 1) or 1-benzosuberone (Eq. 2)
with thiourea in the presence of iodine at 100 °C generated the tri-
cyclic thiazoleamines 36 and 32, respectively, which upon neutral-
ization served as starting materials for the subsequent steps.
Thiazoleamine 37 (Scheme 1, Eq. 1) was prepared by forming the
Schiff base followed by reduction using sodium cyanoborohydride.
Thiazoleamine 32 was treated with 2,6-difluorobenzoyl chloride in
triethylamine containing dichloromethane to obtain the
corresponding benzamide derivative 43 (Scheme 1, Eq. 2). Thiaz-
oleamine 45 (Table 2) was obtained by alkylating amine 30 with
6-azidohex-1-yl tosylate following the procedure of Salvatore
et al.10 Benzamides 46 and 47 (Table 2) were synthesized by cou-
pling thiazoleamine 30 and 32, respectively, with 4-propynyloxy-
benzoic acid (see Supplementary data).
A
collection of 35 tricyclic thiazole derivatives (Table 1)
comprising of thiazoleamines and their amides were synthesized
and screened for their antiproliferative activities in HUVEC culture.
Among a series of 4H,5H-naphto[1,2-d]thiazoleamines containing
different patterns of methoxy substitutions on the A-ring (see Eq.
1 in Scheme 1 for ring designation), all the parent primary amines
1, 4, 6, 10, and 12 failed to register an IC50 below 10
single furanyl substituted thiazoleamine 7 showed a moderate
inhibition of HUVEC (4.5 M). We had acetamide, propanamide,
lM. Only a
20
lM) either isoforms of human MetAPs (hMetAP1 and hMetAP2),
l
suggesting that HUVEC inhibition proceeded through a different
mechanism. Herein, we disclose a structure–activity relationship
(SAR) study of this novel class of endothelial cell inhibitors.
The tricyclic thiazoles were synthesized as per our earlier
procedure7 using a variation of Hantzsch thiazole synthesis. Two
and hexanamide derivatives in this series where both the hexana-
mides 3 and 9 inhibited HUVEC proliferation moderately (3.0 and
3.7
(4.2
l
l
M, respectively), but none of the acetamides except 11
M) exhibited HUVEC inhibition. Compounds 14 and 15, com-
prising of an inversely fused tricyclic thiazole ring system, were
ineffective. Next in our SAR effort, we produced and screened thi-
azoles embodying a contracted B-ring (16–19), a completely sev-
ered B-ring (20–23), and a totally eliminated A-ring (24 and 25).
⇑
Corresponding author. Tel.: +1 410 955 4619.
E-mail address: joliu@jhu.edu (J.O. Liu).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.067

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S. Bhat et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2733–2737
Table 1
Inhibition of HUVEC proliferation by thiazoles
Entry
Structure
Substituents
HUVECa
(IC50, lM)
(if not specified, R# = H)
1
2
3
4
5
6
7
8
9
10
11
12
13
R2 = H
>10
>10
3.0 1.0
>10
>10
>10
4.5 1.2
>10
3.7 1.0
>10
4.2 1.1
>10
>10
R2 = COMe
R2 = CO(CH2)4Me
R
2 = H, R6 = OMe
R2 = COMe, R6 = OMe
R2 = H, R7 = OMe
R2 = CH2(2-furanyl), R7 = OMe
R2 = COMe, R7 = OMe
R2 = CO(CH2)4Me, R7 = OMe
R2 = H, R8 = OMe
R2 = COMe, R8 = OMe
R2 = H, R7, R8 = OMe
R2 = COEt, R7, R8 = OMe
14
15
R2 = H
>10
>10
R
2 = COMe
16
17
18
19
R2 = H, R6 = OMe
>10
4.4 1.5
>10
>10
R2 = COMe, R6 = OMe
R2 = H, R6 = F
R2 = COEt, R6 = F
20
21
22
23
R2 = H, R6, R7 = OMe
>10
>10
>10
>10
R
2 = COMe, R6, R7 = OMe
R2 = H, R7 = Cl
R2 = COMe, R7 = Cl
24
25
R2 = H
R
>10
>10
2 = COMe
26
27
28
29
30
31
R2 = H, X = O
R
>10
>10
>10
>10
1.9 0.3
2.0 0.3
2 = COEt, X = O
R2 = H, X = O, R8 = F
R2 = COEt, X = O, R8 = F
R2 = H, X = S
R2 = COEt, X = S
32
33
34
35
R1, R2 = H, X = CH2
>10
3.7 1.1
1.5 0.3
5.1 1.1
R
1 = H, R2 = COEt, X = CH2
R1, R2 = COEt, X = CH2
R1, R2 = H, X = O, R8–R9 = OCH2O
a
IC50 values are averages of three independent experiments and the standard deviation is given.
Scheme 1. A typical synthesis of tricyclic thiazole derivatives. Reagents and conditions: (i) thiourea, iodine, EtOH, 100 °C, 3 h, then aqueous NaHCO3; (ii) furfural/MgSO4/
MeOH and then NaBH3CN; (iii) 2,6-difluorobenzoyl chloride, Et3N/DCM.
None of these compounds (16–25) except N-(6-methoxy-8H-inde-
no[1,2-d]thiazol-2-yl)acetamide (17, 4.4 M) exhibited HUVEC
inhibition. We then introduced an oxygen atom in the B-ring to ob-
inactive. Finally, when we prepared and tested thiochromenothiaz-
ole derivatives 30 and 31, both the parent thiazoleamine and its
propanamide were found to inhibit HUVEC proliferation with an
l
tain chromenothiazole derivatives 26 through 29, but they were all
IC50 around 2 lM. Another variation we tried was to enlarge the

S. Bhat et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2733–2737
2735
Table 2
Two classes of thiazoles: antiproliferative activities against four cell lines (IC50
,
lM)
Entry
Structure
Substituents (if not specified, R# = H)
HUVECa
Jurkat-Ta
BT474a
HeLaa
Selectivity indexb
30
31
H
COEt
1.9 0.3
2.0 0.3
0.9 0.1
4.9 0.5
8.2 1.4
5.9 1.1
6.8 1.1
8.0 1.1
0.5/4.3/3.6
2.5/3.0/4.0
41
45
46
COPh(2,6-difluoro)
(CH2)6N3
COPh(4-OCH2C„CH)
12.0 2.2
5.9 1.1
2.9 0.8
11.8 1.1
ND
4.6 0.9
7.2 1.3
ND
0.3 0.2
13.1 2.1
ND
5.0 1.1
—
—
1.6/0.1/1.7
36
37
H
1.9 0.4
1.1 0.3
1.6 0.7
2.5 0.6
3.8 1.1
2.3 0.8
11.9 1.0
10.3 1.1
0.8/2.0/6.3
2.3/2.1/9.4
CH2(2-furanyl)
38
42
CO2Et
COPh(2,6-difluoro)
2.8 0.4
5.2 0.4
6.3 1.0
17.3 1.1
6.2 1.1
3.6 1.1
6.5 1.6
>20
2.3/2.2/2.3
3.3/0.7/>4
32
33
34
39
43
47
R1, R2 = H
19.1 1.2
3.7 1.1
1.5 0.3
9.2 1.2
1.5 0.4
7.3 1.1
>20
>20
2.8 0.4
>20
>20
ND
>20
>20
>20
>20
>20
ND
>20
>20
5.9 1.1
>20
15.2 2.2
ND
—
R
2 = COEt
>5/>5/>5
1.9/>13/4
—
>13/>13/10
—
R1, R2 = COEt
R2 = CO2Et
R2 = COPh(2,6-difluoro)
R2 = COPh(4-OCH2C„CH)
40
44
H
14.8 1.2
13.5 2.1
>20
>20
>20
>20
16.2 2.3
>20
—
—
COPh(2,6-difluoro)
a
IC50 values are averages of three independent experiments and the standard deviation is given.
Selectivity index: IC50 values of Jurkat-T/BT474/HeLa compared to that of HUVEC. ND: not determined.
b
B-ring to make seven-membered tricyclic thiazole derivatives 32
through 35 and in this set all of them exhibited IC50 values below
10 lM, except thiazoleamine 32. Reading into the SAR data accu-
collection of compounds. The potency of HUVEC inhibition of ethyl
carbamate 38 was close to that of propanamide 31. In the series of
benzocycloheptathiazoles, imide 34 turned out to be more potent
mulated so far, we reasoned that the important feature contribut-
ing to the antiproliferative activity of the thiazoleamines against
HUVEC is the tricyclic framework (chiefly based on the fact that
compounds 20 through 25 were inactive). We also concluded that
the amino group of tricyclic thiazoleamine is permissive to deriva-
tization without signigicant loss in activity (IC50 s of amine 30 and
amides 3, 9, 31, 33, and 34 are below 4 lM). Interestingly, further
analysis revealed that the two structural scaffolds with good HU-
VEC inhibitory activity, namely thiochromenothiazole and benzo-
cycloheptathiazole are perfectly superimposable ( Fig. 1) due to
the larger atomic radius of sulfur in 31 spanning the space occu-
pied by two methylene groups in the seven-membered ring coun-
terpart 33. Thus, we reckoned that further medicinal chemistry
based upon thiochromenothiazole and benzocycloheptathiazole
skeletons is likely to afford more potent inhibitors of HUVEC pro-
liferation. We synthesized and evaluated 8-chloro substituted thio-
chromeno[1,2-d]thiazoleamine 36 which was found to be about as
potent as the parent thiazoleamine 30 for inhibiting the growth of
HUVEC (Table 2). Interestingly, when thiazoleamine 36 was alkyl-
ated with 2-furanylmethyl group, the resulting secondary amine
37 became the most potent inhibitor of HUVEC in the entire
for HUVEC inhibition than the amide 33 (1.5 vs 3.7 lM). However,
carbamate 39 was an inferior inhibitor. Remarkably, in the series of
thiochromenothiazoles both amine (30, 36, and 37) and amide (31
and 38) derivatives were generally potent at inhibiting HUVEC pro-
liferation, whereas in the case of benzocycloheptathiazoles—32
through 34, 39, and 40—strictly the amide 33 and imide 34 were
good inhibitors, but the parent amines 32 and 40 were poor inhib-
itors. At this juncture it was deemed necessary to assess if the
HUVEC inhibiting thiochromenothiazoles and benzocyclohepta-
thiazoles were capable of exhibiting cell-type selectivity. We
assessed the effects of these compounds on the proliferation of
three cancer cell lines—Jurkat-T (T cell leukemia), BT474 (breast
cancer), and HeLa (cervical cancer). Thiochromenothiazoles 30,
31, and 36 through 38 were in general only twofold more selective
to HUVEC. On the other hand, of the two benzocycloheptathiazole
amides that inhibited HUVEC potently, 33 showed an impressive
selectivity for HUVEC (>fivefold), and 34 was reasonably selective.
Upon a closer scrutiny of the cell inhibition data, we became
curious with the observation that compounds 30, 34, 36, and 37
manifested high inhibitory activities against Jurkat-T cells besides
inhibiting HUVEC. A thorough literature search revealed that ana-
logs of N-(4H,5H,6H-benzo[6,7]cyclohepta[2,1-d]thiazol-2-yl)2,6-
difluorobenzamide and the corresponding inversely fused thiazole
derivatives were claimed to be potential immunosuppressive
agents.11 Thus, we synthesized few more analogs consisting of
2,6-difluorobenzamide moiety—41 and 42 derived from thiochro-
menothiazoles, and 43 and 44 derived from benzocycloheptathiaz-
oles. Among these benzamides, 43 turned out to be a potent
’
(1.5 lM) and highly selective inhibitor of HUVEC, while inhibition
by 42 was moderate. Surprisingly, benzocycloheptathiazoleamide
44 was nearly ten times less potent than 43 and this can only be
attributed to the presence of a fluoro group on the A-ring.
Analyzing the SAR of thiochromenothiazole and benzocyclo-
heptathiazole derivatives thus far (Table 2), we concluded that
the A-ring and the 2-amino group on the thiazole are the two
apparent loci amenable for modifications to seek potency improve-
ment and perhaps attachment of affinity probes. Thus, we prepared
two thiochromenothiazoleamine derivatives 45 and 46, and a
Figure 1. Structures of 31 (green) and 33 (gray) were minimized (MM2) and were
overlayed using Chem3D.

2736
S. Bhat et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2733–2737
Control (DMSO)
37 (2 M)
37 (5 M)
37 (10 M)
43 (2 M)
43 (5 M)
43 (10 M)
Figure 2. Representative images of tubes formed on Matrigel after seeding with HUVEC and treating with DMSO, 37 or 43 (2, 5, and 10 lM). Tubes were stained with Calcein-
AM and observed under fluorescence microscope at 40Â magnification. Tube formation was quantified using AngioQuant v1.33 software (The MathWorks, Natick, MA). The
experiments were performed in triplicate.
benzocycloheptathiazoleamide 47, all carrying probe attachable
functionalities (via azide-alkyne Huisgen cycloaddition). Not only
did these derivatives retain HUVEC inhibitory activity, but also,
amide 46 which is a 2.9 lM inhibitor, may serve as a useful tool
in the target identification studies.
thiazoles emerged as the most potent inhibitors. Two most potent
HUVEC inhibitors 37 and 43 (1.1 and 1.5 M respectively) repre-
l
senting the two tricyclic scaffolds stated above, also inhibited
endothelial tube formation. Also, benzocycloheptathiazole 43 is a
very selective inhibitor of HUVEC and hence these compounds
can serve as potential leads for the development of promising anti-
angiogenic agents. We will pursue synthesis of more analogs re-
lated to these two lead structures to seek further improvement
in potency and we plan to undertake a study to elucidate the
mechanism of HUVEC inhibition by thiochromenothiazoles and
benzocycloheptathiazoles.
In order to evaluate the effectiveness of these two classes of
compounds in blocking angiogenesis, we performed in vitro tube
formation assay12 with a potent thiochromenothiazoleamine 37
and a selectively potent benzocycloheptathiazole derivative 43 (
Fig. 2). The HUVEC tube formation was inhibited by both 37 and
43 in a dose-dependent manner. An aggregate of tube length, size,
and number of junctions was quantified (Fig. 2). Thiazoleamine 37
inhibited HUVEC tube formation very potently (ꢀ35% of control at
Acknowledgment
5
l
M) whereas 43 showed a moderate inhibition (ꢀ75% of control
This work was supported in part by NIH (R01 CA078743).
at 5 M).
l
In conclusion, we identified tricyclic thiazole derivatives as
inhibitors of HUVEC proliferation with distinct structures and
new mechanisms. We prepared, in total, 47 analogs by systemati-
cally manipulating the three rings of the tricyclic thiazoles. Two
tricyclic systems, thiochromenothiazoles and benzocyclohepta-
Supplementary data
Supplementary data (synthesis and analytical data for the tricy-
clic thiazoles, protocols for cell proliferation assays, representative

S. Bhat et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2733–2737
2737
5. Motzer, R. J.; Hutson, T. E.; Tomczak, P.; Michaelson, M. D.; Bukowski, R. M.;
Rixe, O.; Oudard, S.; Negrier, S.; Szczylik, C.; Kim, S. T.; Chen, I.; Bycott, P. W.;
Baum, C. M.; Figlin, R. A. N. Engl. J. Med. 2007, 356, 115.
6. Loges, S.; Schmidt, T.; Carmeliet, P. Genes Cancer 2010, 1, 12.
7. Bhat, S.; Olaleye, O.; Meyer, K. J.; Shi, W.; Zhang, Y.; Liu, J. O. Bioorg. Med. Chem.
2012, 20, 4507.
dose–response curves of HUVEC inhibition, and protocol for HU-
VEC tube formation assay) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.
2013.02.067.
8. Griffith, E. C.; Su, Z.; Turk, B. E.; Chen, S.; Chang, Y.; Wu, Z.; Biemann, K.; Liu, J.
Chem. Biol. 1997, 4, 461.
References and notes
9. Sin, N.; Meng, L.; Wang, M. Q.; Wen, J. J.; Bornmann, W. G.; Crews, C. M. Proc.
Natl. Acad. Sci. U.S.A. 1997, 94, 6099.
1. Abdollahi, A.; Folkman, J. Drug Resist. Updat. 2010, 13, 16.
2. Ebos, J. M.; Kerbel, R. S. Nat. Rev. Clin. Oncol. 2011, 8, 210.
3. Hanahan, D.; Coussens, L. M. Cancer Cell 2012, 21, 309.
4. Hurwitz, H.; Fehrenbacher, L.; Novotny, W.; Cartwright, T.; Hainsworth, J.;
Heim, W.; Berlin, J.; Baron, A.; Griffing, S.; Holmgren, E.; Ferrara, N.; Fyfe, G.;
Rogers, B.; Ross, R.; Kabbinavar, F. N. Engl. J. Med. 2004, 350, 2335.
10. Salvatore, R. N.; Nagle, A. S.; Jung, K. W. J. Org. Chem. 2002, 67, 674.
11. Che, Q.; Vo, N. H.; Chen, S.; Sun, L. World Patent 089305, 2009.
12. Shim, J. S.; Matsui, Y.; Bhat, S.; Nacev, B. A.; Xu, J.; Bhang, H. C.; Dhara, S.; Han,
K. C.; Chong, C. R.; Pomper, M. G.; So, A.; Liu, J. O. J. Natl. Cancer Inst. 1855, 2010,
102.