Furanyl-1,3-thiazol-2-yl and benzoxazol-5-yl acetic acid derivatives: Novel classes of heparanase inhibitor

Article abstract of DOI:10.1016/j.bmcl.2005.03.014

Using a furanylthiazole acetic acid as a starting point, a novel series of benzoxazol-5-yl acetic acid derivatives have been identified as heparanase inhibitors. Several compounds possess an IC₅₀ of ～200 nM against heparanase, for example, tran

Full text of DOI:10.1016/j.bmcl.2005.03.014

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2295–2299
Furanyl-1,3-thiazol-2-yl and benzoxazol-5-yl acetic acid
derivatives: novel classes of heparanase inhibitor
Stephen M. Courtney,a, Philip A. Hay,a, Richard T. Buck,a, Claire S. Colville,a
David J. Phillips,a David I. C. Scopes,a Faye C. Pollard,a Martin J. Page,b,à
James M. Bennett,b Margaret L. Hircock,b Edward A. McKenzie,b,§ Maina Bhaman,b
Robert Felix,b Colin R. Stubberfieldb,* and Paul R. Turnerb
aDepartment of Medicinal Chemistry, Celltech R&D Ltd, 4-10 The Quadrant, Abingdon Science Park, Abingdon,
Oxfordshire OX14 4YS, UK
bDepartment of Oncology, Celltech R&D Ltd, 4-10 The Quadrant, Abingdon Science Park, Abingdon,
Oxfordshire OX14 4YS, UK
Received 1 July 2004; revised 2 March 2005; accepted 3 March 2005
Abstract—Using a furanylthiazole acetic acid as a starting point, a novel series of benzoxazol-5-yl acetic acid derivatives have been
identified as heparanase inhibitors. Several compounds possess an IC50 of ꢀ200 nM against heparanase, for example, trans 2-[4-[3-
(3,4-dichlorophenylamino)-3-oxo-1-propenyl]-2-fluorophenyl]benzoxazol-5-yl acetic acid (16e). Several of the compounds show
anti-angiogenic properties. Improvement to the DMPK profile of compounds has provided compounds of potential use in in vivo
models.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Heparanase, an endo-b-glucuronidase that cleaves hepa-
ran sulfate, is implicated in several physiological and
pathological processes.1,2 It contributes to the remodel-
ling of the extracellular matrix and basement mem-
branes, prerequisite for angiogenesis and egression of
metastatic tumour cells; its expression correlating with
the metastatic potential of tumour cells and with angio-
genesis.3–7 Although these data promote heparanase as
an attractive drug target, progress in this area has been
limited by the lack of small molecule inhibitors. The
most advanced inhibitor PI-88,8 which has as its princi-
pal component a highly sulfated pentasaccharide, is cur-
rently in Phase II clinical trials. The challenge, therefore,
is to find small, drug-like molecules with acceptable
DMPK properties for evaluation in animal models
and as therapeutic leads.
We have recently reported on a series of 2,3-dihydro-
1,3-dioxo-1H-isoindole-5-carboxylic acids as heparan-
ase inhibitors, exemplified by (1), which has an IC50 of
200 nM.9 In a further phase to our research in this area,
we discovered the furanylthiazole acetic acid (2) to be a
heparanase inhibitor (IC50: 25 lM). Although
a
relatively weak starting point for a medicinal chemistry
programme, the paucity of reported heparanase inhibi-
tors, coupled with its scope for structural changes,
NHCH2CH2Me
O
O
N
N
Keywords: Heparanase; Angiogenesis; Metastasis; b-glucuronidase;
Benzoxazole.
O
HO2C
*
Cl
Corresponding author. Tel.: +44 1235 543291; e-mail: colin.
stubberfield@celltechgroup.com
(1)
Present address: Evotec OAI, 151 Milton Park, Abingdon, Oxford-
shire OX14 4SD, UK.
Cl
N
à Present address: OSI Pharmaceuticals, Watlington Road, Oxford
OX4 6LT, UK.
O
CO2H
Cl
S
§ Present address: School of Biological Sciences, University of Man-
chester, Manchester M13 9PT, UK.
(2)
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.014

2296
S. M. Courtney et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2295–2299
O
O2N
Cl
R1
H
N
S
Cl
a, b, c
O
d, e, f
S
O
Me
CO2Me
R2
N
O
O
CO2H
N
(3)
(4a-h, 5a-e)
Scheme 1. Reagents and conditions: (a) 2-chloro-4-nitroaniline, NaNO2, CuCl2, aq HCl, 37%; (b) TBABr3, AcOH, CH2Cl2, 56%; (c)
H2NC(@S)CH2CO2Me, i-PrOH, 68%; (d) Zn, AcOH, EtOH, 99%; (e) ArCOCl, pyridine; (f) NaOH, H2O, MeOH.
Table 2. In vitro inhibition of heparanase and anti-angiogenic activity
of (5a–e)
encouraged us to explore the SAR of this scaffold. Since
it was established early on that the 2-chloro substituent
appeared critical for retaining inhibitory activity, initial
modifications focussed on replacing the 4-chloro substi-
tuent. Specifically, introduction of an amino function al-
lowed the introduction of a range of groups via parallel
synthesis.
Cl
O
N
O
N
R1
R2
CO2H
H
S
Compound
R1
R2
Heparanase
inhibitiona
IC50, lM
Angiogenesis
inhibitiona
IC50, lM
The synthesis of these analogues centred on a diazotisa-
tion reaction of 2-chloro-4-nitroaniline on 2-acetyl
furan, with subsequent a-bromination and thiazole ring
formation providing the key intermediate (3). It was
then a straightforward matter to reduce the nitro group
and introduce selected amides to provide (4a–h) and
(5a–e) (Scheme 1). Compounds were evaluated for their
heparanase inhibitory activity and their ability to inhibit
angiogenesis as previously described.9 A modified benz-
amide moiety resulted in approximately an order of
magnitude increase in potency, for example (4b), which
had an IC50 of 2.5 lM against heparanase and apprecia-
ble activity (IC50: 2 lM) in inhibiting angiogenesis
(Table 1). However, a brief survey of substituent
changes on the benzamide moiety revealed only a mod-
est impact on potency.
5a
5b
5c
5d
5e
H
H
4-Br
0.45
0.4
0.3
0.6
0.4
15
1
H
2-Cl
H
4-Cl
4-OCF3
4-MeO
>20
20
5
H
a Mean of at least two determinations; NT: not tested.
better activity in the angiogenesis assay than any of the
other analogues.
Although these data were encouraging, the synthetic ac-
cess to the furanylthiazole acetic acids was often capri-
cious. Furthermore, the best compound to date, (5b),
was poorly bioavailable and had a short half-life
(0.5 h) in mice. Accordingly, replacements for this unit
were sought. Molecular overlays of bicyclic heteroaro-
matic acetic acids with furanylthiazole acetic acids
(e.g., (5b)) suggested that such structural ÔmorphingÕ
might provide an acceptable surrogate for the latter sys-
tem. To validate this approach, the synthesis of two
ÔpilotÕ structures was undertaken: (11i) and its positional
isomer (7).
Extending the amide moiety to a cinnamoyl group re-
sulted in a 10–20-fold increase in in vitro potency
against heparanase, compared to the benzamide series
(Table 2). However, for the relatively small group of
analogues evaluated within the cinnamoyl series there
was little impact of the nature of phenyl ring substitu-
tion on the level of heparanase inhibition. Noteworthy
is the fact that the 4-bromo derivative (5b) showed much
It was pleasing to find that (11i) displayed heparanase
inhibitory activity (IC50: 0.4 lM) comparable with that
Table 1. In vitro inhibition of heparanase and anti-angiogenic activity
of (4a–h)
Cl
O
N
N
HO2C
H
N
O
CO2H
R1
R2
Br
N
H
S
O
Cl
O
Compound
R1
R2
Heparanase
inhibitiona
IC50, lM
Angiogenesis
inhibitiona
IC50, lM
(11i)
Br
4a
4b
4c
4d
4e
4f
H
H
4-Cl
>30
2.5
3.5
3.5
1.5
1.0
8.0
4.0
NT
2.0
H
H
4-CF3
4-Cl
7.5
0.5
H
N
2-Cl
H
4-OCF3
4-Cl
0.5
O
N
HO2C
3-Cl
H
NT
NT
>20
O
4g
4h
4-MeO
H
Cl
3-CN
a Mean of at least two determinations; NT: not tested.
(7)

S. M. Courtney et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2295–2299
2297
R
R
NO2
OH
NH2
OH
a, b
c, d
e, f, g
N
O
HO2C
N
O
MeO2C
MeO2C
HO2C
H
N
NO2
O
Ar
(9)
(10)
(11a-k,12a-p)
Scheme 2. Reagents and conditions: (a) MeOH, HCl, D, 90%; (b) H2, Pd–C, EtOAc, 92%; (c) O2NC6H3(R)COCl, THF; (d) TsOH, toluene, D; (e) Zn,
AcOH, EtOH; (f) ArCH@CHCOCl, NEt3, THF; (g) LiOH, H2O, THF.
for (5b), although its activity in the angiogenesis assay
(IC50: 5 lM) was somewhat less than that for its furanyl-
thiazole counterpart. In contrast, compound (7) was sig-
nificantly less active against heparanase (IC50: 8.5 lM).
These results prompted further exploration of the SAR
around (11i) with the principal aims of improving the
anti-angiogenic activity and the pharmacokinetic pro-
file. The synthesis of this structural class employed a
two-stage coupling/dehydrative methodology for gener-
ating key benzoxazole intermediates (10) from the ami-
nophenol (9), thus allowing ready introduction of
different substituents in the central aromatic ring. It
was then a relatively straightforward matter to reduce
the nitro group, couple the resulting aniline to cinnamic
acid chlorides and finally liberate the free benzoxazole-
acetic acids (11a–j, 12a–p) (Scheme 2).
Table 3. In vitro inhibition of heparanase and anti-angiogenic activity
of (11a–k)
R
2
3
X
Y
N
O
HO2C
H
N
O
Compound
R
X, Y
Heparanase
inhibitiona
IC50, lM
Angiogenesis
inhibitiona
IC50, lM
11a
11b
11c
11d
11e
11f
11g
11h
11i
H
H
3
NT
0.75
0.6
NT
H
4-F2.5
3-Br
4-Br
H
>20
20
H
H
3-MeO
4-MeO
H
2.5
3.0
NT
NT
NT
20
H
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
>30
0.75
0.4
3-Br
4-Br
7.5
NT
5
N
HO2C
H
N
11j
11k
3-MeO
2,4-Di-Cl
2.5
0.35
O
Br
(CH2)n
Cl
O
a Mean of at least two determinations; NT: not tested.
(8a) n = 0
(8b) n = 1
(8c) n = 2
respectively, showed greatly enhanced anti-angiogenic
properties (IC50: ꢀ1 lM) (Table 4). Noteworthy is the
3,4-dichloro analogue (12n), which showed the greatest
potency to date in the angiogenesis assay, in comp-
arison to the corresponding 2,4-dichloro derivative,
which was inactive. The reason for this is not readily
apparent.
We initially probed the nature of the link between the
amide group and the pendant aromatic ring. In fact, this
appears to be critical for the observed activity. Thus, the
p-bromobenzamide (8a) loses much activity (IC50:
>10 lM) and the benzyl (8b) and dihydrocinnamoyl
(8c) analogues had IC50s of 3 lM and 4 lM, respec-
tively. It was thus confirmed that further optimisation
should focus on the cinnamoyl series.
We next examined reversal of the cinnamic acid amide
moiety to provide structures (15a–l) (Table 5). Such
compounds were readily prepared via the route outlined
in Scheme 3. This route centred on a successful Heck
reaction of the 2-(4-bromophenyl)benzoxazoles (13) to
provide the cinnamic acid esters (14). Selective cleavage
of the t-butyl ester followed by amide formation and
saponification afforded the benzoxazole acetic acids
(15a–l) and (16a–g) (Table 6).
It was important to ascertain whether the 2-chloro sub-
stituent in the central aromatic ring was actually con-
tributing to the activity profile to address these two
sets of compounds (11a–e) and (11g–j). The data for
these compounds (Table 3) show that the heparanase
inhibitory activity is essentially unaffected by the intro-
duction of the 2-chloro substituent. From this exercise,
no improvement on the profile of (11i) was achieved in
terms of heparanase inhibition and anti-angiogenic ef-
fects. However, it was the introduction of a 3-fluoro sub-
stituent that had a more significant effect on the latter
aspect.
When the central phenyl ring was unsubstituted, the ob-
served SAR against heparanase was similar to the Ôfor-
wardÕ cinnamic acid amide orientation (cf. (11a, 11c–f)
and (15a–d)). Notably, introducing a fluoro substituent
ortho to the 4-methoxy group of (15d) resulted in a 10-
fold increase in anti-heparanase activity (cf. (15j)). The
4-bromo derivative (15b) showed potent activity in the
angiogenesis assay (IC50: 0.25 lM). However, the most
potent heparanase inhibitors were obtained when a
Thus, although the heparanase inhibitory potency of the
3-fluoro series mirrored their unsubstituted counter-
parts, the 3- and 4- bromo analogues, (12k) and (12d),

2298
S. M. Courtney et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2295–2299
Table 4. In vitro inhibition of heparanase and anti-angiogenic activity
of (12a–n)
Table 5. In vitro inhibition of heparanase activity of (15a–l)
R
X
N
R
HO2C
H
N
2
3
X
Y
N
O
HO2C
H
N
O
Y
O
O
Compound
R
X, Y
Heparanase inhibitiona
IC50, lM
Compound
R
X, Y
Heparanase
inhibitiona
IC50, lM
Angiogenesis
inhibitiona
IC50, lM
15a
15b
15b
15c
15d
15e
15f
15g
15h
15i
H
H
H
H
H
H
H
H
H
H
H
H
H
3-Br
4-Br
0.5
0.7
2-OMe
3-OMe
4-OMe
>10
3.0
3.0
12a
12b
12c
12d
12e
12f
12g
12h
12i
3-FH
3-F4-F
3-F4-Cl
5.5
NT
NT
3.0
NT
2.5
0.4
2-OCHF2
4-OCHF2
2,4-Di-Cl
4-F2.5
8.5
1.5
3-F4-Br
3-F4-CF
3-F4-MeO
3-F4-CN
3-F3-Cl
1
2.0
NT
3
0.4
3.0
0.8
0.7
NT
1
7.5
2-Cl
3.0
0.3
15j
3-F, 4-OMe
3-Cl, 4-OMe
3,4-Di-OMe
3-F,23-F-Cl
0.3
5
2
15k
15l
3.0
12j
3-F3-Cl, 4-F0.75
3-F3-Br
3-F2-Cl
>10
12k
12l
0.8
2.5
0.35
0.8
1
NT
>20
0.2
a Mean of at least two determinations; NT: Not tested.
12m
12n
3-F2,4-Di-Cl
3-F3,4-Di-Cl
a Mean of at least two determinations; NT: not tested.
Table 6. In vitro inhibition of heparanase activity of (16a–g)
R
2
3
X
Y
N
O
HO2C
H
N
2-Fsubstituent was introduced into the central
phenyl ring. Thus, (16a), (16d) and (16e) all displayed
IC50s of 200 nM with (16e) showing moderate activity
in the angiogenesis assay (IC50: 2 lM).
O
Compound
R
X, Y
Heparanase inhibitiona
IC50, lM
When administered to mice, both (12d) and (15b)
showed a plasma concentration of ꢀ10x the heparanase
IC50 following oral dosing at 20 mg/kg. Compound
(12d) showed no significant inhibition of the major
human cytochrome P450 isozymes (Cyp3A4 and Cyp2C9,
IC50: >30 lM), had improved aqueous solubility over
the furanylthiazole acetic acids (5b) and displayed mod-
est cell penetration in a Caco2 cell model of intestinal
drug transport10 (A ! B: 0.47 · 106 cm sꢁ1, B ! A:
0.24 · 106 cm sꢁ1).
16a
16b
16c
16d
16e
16f
16g
2-F3-Br
2-F4-Br
0.2
0.4
2-F2,4-Di-F 0.75
2-F2,4-Di-Cl
2-F3,4-Di-Cl
0.2
0.2
2-F, 43-OFMe
2-F4-CN
0.45
0.3
a Mean of at least two determinations; NT: Not tested.
has been made towards identifying compounds, which
should serve as useful biological tools and may also
provide a basis for the design of novel therapeutic
agents.11
In summary, the data presented here describe a series of
benzoxazoles as potent inhibitors of heparanase. The
DMPK profile of some of these suggests that progress
R
R
NH2
OH
N
O
b
N
O
a
MeO2C
MeO2C
MeO2C
Br
CO2tBu
(13)
(14)
R
R
c
d, e, f
N
O
N
O
HO2C
MeO2C
O
CO2H
NH
X
Y
(15a-l)
Scheme 3. Reagents and conditions: (a) BrC6H3(R)COCl, dioxane, lW 210 ꢁC; (b) H2C@CHCO2t-Bu, Pd(OAc)2, o-Tol3P, NEt3, DMF, lW 150 ꢁC;
(c) TFA, CH2Cl2; (d) (COCl)2, DMF, THF; (e) BrArNH2, NEt3, THF; (f) LiOH, H2O, THF.

S. M. Courtney et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2295–2299
2299
6. Vlodavsky, I.; Goldshmidt, O.; Zcharia, E.; Metzger, S.;
Chajek-Shaul, T.; Atzmon, R.; Guatta-Rangini, Z.; Fre-
idmann, Y. Biochimie 2001, 83, 831.
7. Vlodavsky, I.; Freidmann, Y. J. Clin. Invest. 2001, 108,
341.
8. Parish, C. R.; Freeman, C.; Brown, K. J.; Francis, D. J.;
Cowden, W. B. Cancer Res. 1999, 59, 3433.
9. Courtney, S. M.; Hay, P. A.; Buck, R. T.; Colville, C. S.;
Porter, D. W.; Scopes, D. I. C.; Pollard, F. C.; Page, M. J.;
Bennett, J. M.; Hircock, M. L.; McKenzie, E. A.;
Stubberfield, C. R.; Turner, P. R. Bioorg. Med. Chem.
Lett. 2004, 14, 3269.
References and notes
1. Parish, C. R.; Freeman, C.; Hulett, M. D. Biochim.
Biophys. Acta 2001, 1471, M99.
2. Fairbanks, M. B.; Mildner, A. M.; Leone, J. W.; Cavey,
G. S.; Mathews, W. R.; Drong, R. F.; Slightom, J. I.;
Bienkowski, M. J.; Smith, C. W.; Bannow, C. A.;
Heinrikson, R. L. J. Biol. Chem. 1999, 274, 29587.
3. Vlodavsky, I.; Freidmann, Y.; Elkin, M.; Aingorn, H.;
Atzmon, R.; Ishai-Michaeli, R.; Bitan, M.; Pappo, O.;
Peretz, T.; Michal, I.; Spector, L.; Pecker, I. Nature Med.
1999, 5, 793.
10. Balimane, P. V.; Chong, S.; Morrison, R. A. J. Pharmacol.
Toxicol. Methods 2000, 44, 301.
4. Hulett, M. D.; Freeman, C.; Hamdorf, B. J.; Baker, R. T.;
Harris, M. J.; Parish, C. R. Nature Med. 1999, 5, 803.
5. Toyoshima, M.; Nakajima, M. J. Biol. Chem. 1999, 274,
24153.
11. All final products were characterised by 1H NMR and
MS.