Synthesis and biological evaluation of norcantharidin analogues: Towards PP1 selectivity

Article abstract of DOI:10.1016/j.bmc.2007.08.028

Simple modifications to the anhydride moiety of norcantharidin have lead to the development of a series of analogues displaying modest PP1 inhibition (low μM IC₅₀s) comparable to that of norcantharidin (PP1 IC₅₀ = 10.3 ± 1.37 μM). However, unlike norcantharidin, which is a potent inhibitor of PP2A (IC₅₀ = 2.69 ± 1.37 μM), these analogues show reduced PP2A inhibitory action resulting in the development of selective PP1 inhibitory compounds. Data indicates that the introduction of two ortho-disposed substituents on an aromatic ring, or para-substituent favours PP1 inhibition over PP2A inhibition. Introduction of a p-morphilinoaniline substituent, 35, affords an inhibitor displaying PP1 IC₅₀ = 6.5 ± 2.3 μM; and PP2A IC₅₀ = 7.9 ± 0.82 μM (PP1/PP2A = 0.82); and a 2,4,6-trimethylaniline, 23, displaying PP1 IC₅₀ = 48 ± 9; and PP2A IC₅ 85 ± 3 μM (PP1/PP2A = 0.56). The latter shows a 7-fold improvement in PP1 versus PP2A selectivity when compared with norcantharidin. Subsequent analysis of 23 and 35 as potential PP2B inhibitors revealed modest inhibition with IC₅₀s of 89 ± 6 and 42 ± 3 μM, respectively, and returned with PP1/PP2B selectivities of 0.54 and 0.15. Thus, these analogues are the simplest and most selective PP1 inhibitors retaining potency reported to date. Crown Copyright

Full text of DOI:10.1016/j.bmc.2007.08.028

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 15 (2007) 7301–7310
Synthesis and biological evaluation of norcantharidin
analogues: Towards PP1 selectivity
Scott G. Stewart,a Timothy A. Hill,a Jayne Gilbert,b Stephen P. Ackland,b
Jennette A. Sakoffb and Adam McCluskeya,*
aChemistry Building, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia
bDepartment of Medical Oncology, Newcastle Mater Misericordiae Hospital, Waratah, NSW 2298, Australia
Received 5 February 2007; revised 17 August 2007; accepted 20 August 2007
Available online 24 August 2007
Abstract—Simple modifications to the anhydride moiety of norcantharidin have lead to the development of a series of analogues
displaying modest PP1 inhibition (low lM IC50s) comparable to that of norcantharidin (PP1 IC50 = 10.3 1.37 lM). However,
unlike norcantharidin, which is a potent inhibitor of PP2A (IC50 = 2.69 1.37 lM), these analogues show reduced PP2A inhibitory
action resulting in the development of selective PP1 inhibitory compounds. Data indicates that the introduction of two ortho-dis-
posed substituents on an aromatic ring, or para-substituent favours PP1 inhibition over PP2A inhibition. Introduction of a p-mor-
philinoaniline substituent, 35, affords an inhibitor displaying PP1 IC50 = 6.5 2.3 lM; and PP2A IC50 = 7.9 0.82 lM (PP1/
PP2A = 0.82); and a 2,4,6-trimethylaniline, 23, displaying PP1 IC50 = 48 9; and PP2A IC5 85 3 lM (PP1/PP2A = 0.56). The lat-
ter shows a 7-fold improvement in PP1 versus PP2A selectivity when compared with norcantharidin. Subsequent analysis of 23 and
35 as potential PP2B inhibitors revealed modest inhibition with IC50s of 89 6 and 42 3 lM, respectively, and returned with PP1/
PP2B selectivities of 0.54 and 0.15. Thus, these analogues are the simplest and most selective PP1 inhibitors retaining potency
reported to date.
Crown copyright ꢀ 2007 Published by Elsevier Ltd. All rights reserved.
1. Introduction
Both PP1 and PP2A are strongly inhibited by a number
of naturally occurring toxins: okadaic acid (1) (PP1
IC50 = 60 nM; PP2A IC50 = 1 nM);10 calyculin A (2)
(PP1 IC50 = 0.5–1.0 nM; PP2A IC50 = 2.0 nM);11 micro-
The intricately linked protein phosphorylation and
dephosphorylation cycle is regulated by a fine balance
between kinases and the phosphatases, respectively.1–6
The prior notion that phosphatases existed merely as
on–off switches moderating the action of kinases is a
vast oversimplification of their role, especially in the cell
cycle.7–9 Further to this, it is now known that phospha-
tases have a vital and delicate role to play in a myriad of
biological processes.7 Within the phosphatase family,
the serine/threonine phosphatases comprising of PP1,
PP2A, PP2B, PP4, PP5, PP6 and PP7 are responsible
for the vast majority of dephosphorylation events in liv-
ing organisms. Of these phosphatases, PP1 and PP2A
account for >90% of phosphatase activity and as such
have received the greatest attention.7–9
cystin-LR (3) (PP1 IC50 = 1.7 nM; PP2A IC50
=
0.04 nM);12 tautomycin (4) (PP1 IC50 = 0.3 nM; PP2A
IC50 = 1 nM);13 tautomycetin (5) (PP1 IC50 = 1.6 nM;
PP2A IC50 = 62 nM)14 and fostriecin (6) (PP1 IC50
=
131,000 nM; PP2A IC50 = 3.4 nM)15 are exemplars of
these toxins. Of these only fostriecin exhibits noteworthy
PP2A selectivity (ꢀ40,000-fold). PP1 selectivity has
proved more problematic to achieve with poor PP1
selectivity reported for tautomycin (ꢀ4-fold), tautomy-
cetin (ꢀ40-fold), a modified microcystin-LA analogue
developed by Chamberlin (ꢀ7-fold)16 and phospatidic
acid (>100-fold).17
The discovery of a selective PP1 inhibitor has the poten-
tial to initiate a number of biochemical studies as to this
protein’s exact role. However, the advent of tautomyce-
tin and phosphatidic acid has resulted in only modest
progress thus far. As with a number of members of
the okadaic acid class of compounds, tautomycetin
displays antibiotic properties,18 has been used as an
Keywords: Cantharidin; Norcantharidin; Small molecule protein phos-
phatase inhibitors; PP1; PP2A; PP2B.
*
Corresponding author. Tel.: +61 249 216486; fax: +61 249
215472; e-mail: Adam.McCluskey@newcastle.edu.au
0968-0896/$ - see front matter Crown copyright ꢀ 2007 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.08.028

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S. G. Stewart et al. / Bioorg. Med. Chem. 15 (2007) 7301–7310
immunosuppressive agent19,20 and induces morphologi-
cal changes (bleb formation) in human leukaemia cells
(K562).21 Studies with tautomycetin have highlighted a
role for PP1 as a positive regulator of Raf-1.22 The ob-
served growth inhibition by tautomycetin in a range of
colorectal cancer cell lines (HT-15, HT-29 and DLD-1)
is believed to be acquired via Raf-1 modulation.23 It is
known that tautomycetin has potential application as
an anti-rejection/immunomodulatory agent potentiating
the effects of cyclosporine following organ transplanta-
tion, suggesting a possible role for PP1, and obviously
a potential new drug target for small molecule PP1 spe-
cific inhibitors (Fig. 1).
first true small molecule inhibitors displaying PP1
selectivity.
2. Results and discussion
Our initial synthetic efforts focused on the simple manip-
ulation of 8, a norcantharidin (11) analogue derived
from anhydride ring opening.29
O
N
H
O
CO CH
2
3
OH
O
8
Of all the known inhibitors of PP1 and PP2A, canthar-
idin (7) is structurally the simplest and displays modest
PP2A selectivity (PP1 IC50 = 1.78 lM; PP2A
IC50 = 0.26 lM),24 it has however tolerated little in the
way of structural modifications that impart either im-
proved potency or selectivity. Sodeoka has had excellent
success utilising the norcantharidin (11) framework as a
protein phosphatase inhibitor core in the design and
synthesis of selective PP2B analogues.25,26 Our group
has had very modest success in this area with modified
norcantharidin analogues;27–35 however, in this instance
the improvements in PP2A versus PP1 selectivity were
the result of a loss of both PP1 and PP2A inhibitory
activity, with PP1 potency less affected. Given our past
and continuing interest in elegantly simple modification
of the synthetically amenable PP1 and PP2A inhibitor,
norcantharidin, we have continued our efforts to devel-
op more potent and selective inhibitors of both PP1
and PP2A.27–35 In this paper, we report our latest devel-
opments in this area and take small steps towards the
PP1 IC = 16 2 μM
50
PP2A IC = 7.7 1.3 μM
50
Thus, a series of analogues based on 8 were prepared
in excellent yields via the exo-selective Diels–Alder36,37
addition of furan and maleic anhydride followed by
simple hydrogenation (Scheme 1).34 Treatment of nor-
cantharidin in THF at room temperature with a series
of substituted aromatic amines afforded the corre-
sponding amide-acid analogues 12 in good to excellent
yields (46–90%). Subsequent biological evaluation
against PP1 and PP2A gave rise to the data shown
in Table 1.
Commencing with the parent aniline (12), we note a
modest level of enzyme inhibitory activity when com-
pared with that of norcantharidin 11 and cantharidin
6 (PP1 IC50s of 10.3 1.37; 7.6 0.78 lM and PP2A
OH
O
CH
3
O
OH
O
O
H
H
H CO
3
N
H
O
H C
3
H
O
N
N
OH
HO
H C OH
O
O
O
3
H
H
O
OH CH
3
H
O
HO
HO
H
O
OH CH
3
P
1
O
CN
OH
O
CO H
2
O
H
3
N
OH OH OCH
HN
NH
2
O
OCH
O
3
O
NH
HN
H C
3
CH
3
H
N
H
N
OH
O
OH
O
OH
H
O
O
O
O
O
O
O
CO H
2
H
O
O
OCH
CH
CH
CH
3
3
3
3
O
3
4
HN
NH
2
OH
O
CH OH
3
O
OH
O
O
NaO P
OH
3
O
OH
O
O
O
O
CH
3
CH
O
O
O
3
OH
O
O
5
6
7
Figure 1. Chemical structures of archetypal protein phosphatase 1 and 2 A inhibitors: (1) okadaic acid; (2) calyculin A; (3) microcystin-LR;
(4) tautomycin; (5) tautomycetin; (6) fostrecin and (7) cantharidin.

S. G. Stewart et al. / Bioorg. Med. Chem. 15 (2007) 7301–7310
7303
O
enzyme selectivity, viz the parent is ꢀ3-fold PP2A
selective, as is the norm for norcantharidin ana-
logues.27–35,38–40 However, extension of the aromatic
ring progressively switched the enzyme selectivity in
favour of PP1 (Table 1, entries 2–4). Although this
increase in relative PP1 selectivity is at the cost of PP1
potency, as previously observed,27 we were intrigued
with these observations and were keen to further exam-
ine the requirements for PP1 selectivity. Accordingly we
synthesized analogues lacking the aromatic ring, but
possessing a moderately sized alkyl chain at this point.
These alkyl chains typically possessed a terminal func-
tional group bearing lone pairs of electrons to mimic
the electron rich nature of the aromatic ring. As can
be seen from Table 1 (entry 5), a terminal N,N-dimeth-
ylaminoethyl (16) substituent effectively lost PP2A
inhibition whilst displaying a modest improvement in
PP1 inhibition relative to 15, consequently PP1 selectiv-
ity was again improved. Further elongation of this
chain, N,N-dimethylaminopropyl (17), improved the
inhibition of both PP1 and PP2A but at the cost of
PP1 selectivity. Viewing continued extension of this side
chain as being counterproductive, and most likely
increasing both PP1 and PP2A potency we sought to
encapsulate the terminal dimethyl groups and amine
functionality within an ethylmorpholino moiety (18).
In this instance we were again rewarded with an increase
in both PP1 and PP2A potency while maintaining PP1
selectivity (Table 1, entry 7). While the observed PP1
selectivities are at best modest, they also represent some
of the highest PP1 selectivities reported to date.
O
O
R
i, ii
iii
+
O
O
O
O
O
OH
O
O
O
9
10
11
12-36
Scheme 1. Synthesis of amide-acid analogues of norcantharidin.
Reagents and conditions: (i) rt, 24 h, Et2O; (ii) 4 atm H2, 10% Pd–C,
3 days; (iii) RNH2, THF, rt 16, or RNH2, 60 ꢁC, 16 h.
Table 1. Protein phosphatase 1 and 2A inhibition by amide-acid
analogues 12–18
O
R
O
OH
O
12-18
a
a
PP1 IC50 PP2A IC50 PP1/PP2A
Entry
R
(lM)
(lM)
H
N
1
24.3 2.8
7.7 0.8
3.2
2.9
12
N
H
2
35
25
0
0
12
0
13
3
4
25
95
4
5
1
H
N
Suitably encouraged about the possibility of developing
a family of PP1 selective inhibitors, we again turned
our attention to aromatic amines. In this series of com-
pound we commenced by placing the alkyl chain on
the aromatic ring, that is, we utilised a series of alkyl
substituted anilines as the nucleophiles to open nor-
cantharidin’s, (11), anhydride ring affording 19–35.
Introduction of an ortho-ethyl (19) returned modest
PP1 and PP2A inhibition, but also PP2A selectivity;
however, the corresponding meta-ethyne side chain
(20) induced a modest increase in potency and mar-
ginal PP1 selectivity (Table 2, entry 2). A series of
methyl substituted analogues (21–23) displayed PP1
selectivity only when both ortho-positions were occu-
pied (Table 2, entries 3 and 5). Compound 22 with a
2,3-dimethyl disposition lost both potency and returned
PP2A selectivity suggesting that for PP1 selectivity the
aromatic ring must be moved out of the amide plane or
possess a considerable degree of hindered rotation. In-
deed, molecular modeling analysis (MacSpartanPro,
data not shown) indicates that most PP1 selective ana-
logues all have the phenyl ring twisting such that it is
orthogonally disposed to the amide moiety. Analogue
23 is one of the most selective small molecule PP1
inhibitors yet reported. In attempts to investigate this
possibility further, we sought to bulk up the alkyl
chains examined and turned our attention to tert-butyl
analogues 24 and 25, interestingly neither of these ana-
logues displayed notable PP1 selectivity, nor PP2A
selectivity (Table 2, entries 6 and 7). It therefore ap-
pears that the presence of a bulky side chain in the
14
75 12
0.79
N
H
15
N
5
6
7
67
43
30
7
7
3
>100
57
<0.67
0.75
N
H
16
2
1
N
H
N
17
O
43
0.70
N
N
H
18
a Mean SEM of three experiments conducted in duplicate.
IC50s of 2.69 0.15; 0.99 0.12 lM, respectively).
However, moving the pendant aromatic ring distal to
the amide function caused a decrease in potency for
both enzymes, with the benzyl-(13) and propylphenyl-
(14) substituents essentially equipotent (Table 1, entries
2 and 3), further extension with the butylphenyl-(15)
analogue produced a 4-fold decrease in PP1 potency
and a 7-fold decrease in PP2A potency, relative to 12.
More interestingly, these analogues display a change in

7304
S. G. Stewart et al. / Bioorg. Med. Chem. 15 (2007) 7301–7310
Table 2. Protein phosphatase 1 and 2A inhibition by amide-acid analogues 19–35
O
R
O
OH
O
19-35
Entry
R
PP1 IC50a (lM)
PP2A IC50a (lM)
PP1/PP2A
2.12
1
51
5
24
3
N
H
19
2
3
17 0.7
18 0.9
0.94
0.78
N
H
20
56 10
72
2
N
H
21
4
17 0.7
18 0.9
0.94
N
H
22
5
6
48
10
9
0
85
11
3
1
0.56
0.91
N
H
23
N
H
24
7
8.2
2
8.2 0.7
1
N
H
25
26
8
9
14
11
2
5
15
15
1
2
0.93
0.73
N
H
O
N
H
27
O
10
11
15
10
1
4
16
14
1
1
0.94
0.71
N
H
28
29
S
N
H

S. G. Stewart et al. / Bioorg. Med. Chem. 15 (2007) 7301–7310
7305
Table 2 (continued)
Entry
R
PP1 IC50a (lM)
PP2A IC50a (lM)
PP1/PP2A
12
23
1
33
2
0.70
N
H
S
30
13
14
36
2
39
2
0.92
2.14
N
H
OH
31
15 0.3
7
1.5
OH
N
H
32
F
F
H
N
15
16
26
16
2
3
26
20
2
1
1
33
CF
3
0.8
H
N
34
O
N
17
6.5 2.3
7.9 0.4
0.82
N
H
35
a Mean SEM of three experiments conducted in duplicate.
para position also leads towards parity and marginal
PP1 selectivity; this is further evidenced by the intro-
duction of a 2-naphthyl (26), a para-OnC6H14 (27), a
para-OCH3 (28) and a para-SCH3 (29) substituent, in
which all analogues returned modest PP1 selectivity
(Table 2, entries 8–11). The SCH3 moiety within which
was also introduced as an ortho substituent (30) re-
sulted in a slight loss in potency at both PP1 and
PP2A, but essentially no change in PP1 selectivity (cf.
Table 2, entries 11 and 12).
the related PP2B. In this instance we restricted our
examination to the two parent compounds, cantharidin
(7) and norcantharidin (11), and the two most PP1 selec-
tive analogues generated herein, compounds 23 and 35.
The data are presented in Table 3.
Of the four analogues examined for PP2B inhibition, only
the parent cantharidin displayed essentially no activity
returning an IC50 > 100 lM. Removal of the methyl sub-
stituents in the analogous norcantharidin results in a
modest level of PP2B inhibition (IC50 = 31 1 lM),
and we also note that analogues 23 and 35 are at best
modest inhibitors (IC50s = 89 6 and 42 3 lM, respec-
tively). By virtue of higher levels of PP1 inhibition, 35
also displays the best PP1/PP2B selectivity (0.15).
Attempts to introduce other functionality to the aro-
matic ring was met with mixed success, an ortho-
CH2OH (31) afforded marginal PP1 selectivity, but
the same substituent meta-disposed (32) returned
inherent PP2A selectivity albeit with an improvement
in potency. The introduction of fluorine substituents,
33 and 34 gave two compounds with good potency
and a drift towards PP1 selectivity, most notably with
the para-CF3 substituent; in this latter case the ob-
served selectivity is better than the para-tert-butyl 24,
suggesting that a combination of para-substituents’
bulk and an electron withdrawing group may aid the
introduction of PP1 selectivity. Our explorations in
this area gave rise to the para-morpholino (35) substi-
tuted analogue that displayed excellent PP1 inhibition
(IC50 < 10 lM, Table 2 entry 17) and selectivity simi-
lar to 34.
3. Conclusions
As part of our on-going investigations towards the
development of PP1 and PP2A selective norcantharidin
analogues, we have synthesized a series of norcanthari-
din analogues displaying PP1 selectivity. In the course
of this work, we have modified the inherent selectivity
of our lead compound (12, ꢀ3-fold PP2A selective)
and developed an analogue with slight decrease in PP1
potency, but a 5-fold decrease in PP2A selectivity (22,
ꢀ0.5-fold PP2A selective), hence the increased PP1
selectivity is at the expense only of PP2A potency. As
a result of this study, we have for the first time devel-
oped a series of PP1 selective analogues, representing a
Having determined the relative PP1 versus PP2A selec-
tivity, we felt it prudent to examine selectivity against

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S. G. Stewart et al. / Bioorg. Med. Chem. 15 (2007) 7301–7310
Table 3. Protein phosphatase 1, 2A and 2B inhibition by cantharidin (7), norcantharidin (11) and amide-acid analogues 23 and 35
Compound
PP1 IC50 (lM)
PP2A IC50 (lM)
PP2B IC50 (lM)
PP1/PP2A
PP1/PP2B
>0.07
O
O
O
7.6 0.78
0.99 0.12
>100
7.7
O
7
O
O
O
10.3 1.37
2.69 0.12
31
89
1
6
3.8
0.33
0.54
O
11
O
N
H
OH
O
48
9
85
3
0.56
O
O
23
O
N
N
H
O
6.5 2.3
7.9 0.4
42
3
0.82
0.15
OH
O
35
aMean SEM of three experiments conducted in duplicate.
potentially important advance in the development of
new, novel and selective PP1 inhibitors. Future work
further exploring this PP1 binding domain will be re-
ported in due course. Importantly these analogues also
display for PP1 over PP2B.
pThr-Ile-Arg) was quantified by colorimetric analysis
using the Malachite Green method. PP1, PP2A and sub-
strate used in the assay were 30 mU/well, 0.3 mU/well
and 200 lM, respectively. The reactions were initiated
by addition of substrate (5 lL) to a mixture containing
enzyme (5 lL), reaction buffer (10 lL; 50 mM Tris–
HCl, pH 7.0, 100 lM CaCl2) and inhibitor (10 lL), pro-
ducing a total reaction volume of 30 lL/well and incu-
bated at room temperature for 60 min. Reactions were
halted via addition of a malachite green solution
(50 lL), and the absorbance readings were taken at
650 nm after 10 min development time. Enzyme dilu-
tions were made with buffer containing 20 mM MOPS,
pH 7.5, 0.15 M NaCl, 60 mM 2-mercaptoethanol,
1 mM MgCl2, 2 mM EGTA, 0.1 mM MnCl2, 1 mM
DTT, 10% glycerol and 0.1 mg/mL serum albumin.
For the PP2B assay, para-nitrophenyl phosphate
(pNPP) was used as a substrate. The reaction was initi-
ated by addition of substrate (12 lL, 2.4 mM) to a mix-
ture (8 lL) containing 57 nM PP2B, 50 mM Tris–HCl,
pH 7.0, 100 lM CaCl2, NiCl2, 0.3 mg/mL BSA and
0.25 lM calmodulin, and inhibitor (10 lL), producing
a total reaction volume of 30 lL/well and incubated at
37 ꢁC for 60 min. For each of the enzymatic assays,
the enzyme and inhibitor were preincubated prior to
addition of substrate. Samples were blanked against
wells containing enzyme and buffer only. Initial inhibi-
tor dilutions were made in DMSO and subsequent dilu-
tions were made in distilled deionised H2O. A dose–
response curve of percentage enzyme activity versus
drug concentration was produced from which an IC50
value was calculated indicating the concentration of
drug required to inhibit enzyme activity by 50%. Data
represent the mean ( SEM) IC50 of three independent
experiments performed in duplicate.
4. Experimental
4.1. General
All starting materials were purchased from Aldrich
Chemical Company and Lancaster Synthesis. Solvents
were bulk, and distilled from glass prior to use. 1H
and 13C spectra were recorded on a Bruker Advance
AMX 300 MHz spectrometer at 300.13 and
75.48 MHz, respectively. Chemical shifts are relative to
TMS as internal standard. All compounds returned sat-
isfactory analyses. High resolution mass spectrometry
was conducted at the University of Wollongong, Aus-
tralia. All compounds returned satisfactory micro anal-
ysis and/or high resolution mass spectral data.
4.2. Protein phosphatase inhibition
Assays were performed according to previously reported
PP1, PP2A and PP2B assays.6 Protein phosphatases PP1
(rabbit skeletal muscle), PP2A (human red blood cells)
and PP2B (bovine brain) were purchased from Upstate
Biotechnology (Lake Placid, NY). Protein phosphatase
assays were carried out according to the manufacturer’s
protocol in the presence or absence of an appropriate
concentration of test compound. For PP1 and PP2A, a
serine/threonine assay was used in which free phosphate
ion released from a substrate phosphopeptide (Lys-Arg-

S. G. Stewart et al. / Bioorg. Med. Chem. 15 (2007) 7301–7310
7307
4.3. 3-Phenylcarbamoyl-7-oxabicyclo[2.2.1]heptane-2-car-
boxylic acid (12)
4.7. 3-(2-Dimethylaminoethylcarbamoyl)-7-oxabicy-
clo[2.2.1]heptane-2-carboxylic acid (16)
1H NMR (DMSO-d6): 9.64 (1H, s, NH), 7.50 (1H, d,
J = 7.6 Hz), 7.25 (1H, t, J = 7.6 Hz, 1H), 7.01 (1H, t,
J = 6.7 Hz), 4.76 (s, 1H), 4.62 (1H, d, J = 3.9 Hz), 3.03
(1H, d, J = 9.4 Hz), 2.92 (1H, d, J = 9.4 Hz), 1.48–1.59
(4H, m).
1H NMR: (CDCl3): 7.34 (1H, t, J = 5.5 Hz), 5.00 (1H, d,
J = 4.65 Hz), 4.76 (1H, d, J = 2.1 Hz) 4.67 (1H, d,
J = 4.8 Hz), 3.57 (2H, q, J = 6.3 Hz) 3.07 (1H, d,
J = 9.6 Hz), 2.96 (1H, d, J = 9.6 Hz), 2.43 (2H, t,
J = 6.8 Hz), 2.22 (6H, s) 1.58–1.50 (4H, m).
13C NMR (DMSO-d6): 173.1, 170.1, 140.1, 129.4 (2C),
123.8, 120.1 (2C), 79.6, 77.8, 54.4, 52.6, 29.8, 29.3.
13C NMR: (CDCl3): 172.3, 170.3, 78.7, 76.7, 53.0, 51.4,
49.4. 44.8, 36.3, 28.8, 28.3.
MP: 170 ꢁC.
MP: 45–47 ꢁC.
HRMS (M+H) calcd 262.1079, found 262.1085.
HRMS (M+H) calcd 257.1501, found 257.1509.
4.8. 3-(3-Dimethylaminopropylcarbamoyl)-7-oxabicy-
clo[2.2.1]heptane-2-carboxylic acid (17)
4.4. 3-Benzylcarbamoyl-7-oxabicyclo[2.2.1]heptane-2-car-
boxylic acid (13)
1H NMR: (CDCl3): 7.34 (1H, t, J = 5.5 Hz), 5.02 (1H, d,
J = 4.5 Hz), 4.80 (1H, d, J = 2.0 Hz) 4.70 (1H, d,
J = 4.2 Hz), 3.57 (2H, q, J = 7.4 Hz), 3.10 (1H, d,
J = 9.6 Hz), 2.96 (1H, d, J = 9.6 Hz), 2.60 (2H, t,
J = 6.7 Hz), 2.22 (6H, s), 1.66 (2H, quin, J = 7.4 Hz),
1.58–1.50 (4H, m).
1H NMR: (DMSO-d6): 7.93 (1H, t, J = 5.7Hz),
7.29–7.21 (5H, m), 4.74 (1H, d J = 2.4 Hz), 4.50
(1H, d J = 2.4 Hz), 4.21 (2H, sept, J = 6.1Hz), 2.92
(1H, d, J = 9.7Hz), 2.84 (1H, d, J = 9.6Hz), 1.56–
1.43 (4H, m).
13C NMR: (DMSO-d6): 172.3, 170.5, 139.4, 128.1, 127.1,
126.6, 78.78, 76.70, 52.9, 51.2, 42.1, 28.9, 28.3.
13C NMR: (CDCl3): 175.5, 174.3, 78.5, 76.3, 53.0, 51.2,
49.2. 44.7, 36.7, 28.5, 28.1, 24.9.
MP: 162–163 ꢁC.
MP: 42–44 ꢁC.
HRMS (M+H) calcd 276.1236, found 276.1238.
HRMS (M+H) calcd 271.1658, found 271.1666.
4.5. 3-(3-Phenylpropylcarbamoyl)-7-oxabicyclo[2.2.1]hep-
tane-2-carboxylic acid (14)
4.9. 3-(2-Morpholin-4-ylethylcarbamoyl)-7-oxabicy-
clo[2.2.1]heptane-2-carboxylic acid (18)
1H NMR: (DMSO-d6): 7.34 (1H, t, J = 5.3Hz), 7.15–
7.28 (5H, m), 4.71 (1H, d, J = 2.3Hz), 4.47 (1H, d,
J = 4.3Hz), 2.98 (2H, q, J = 6.2 Hz), 2.52 (2H, t,
J = 7.5Hz), 1.64 (2H, quin, J = 7.3Hz), 1.43–1.56
(4H, m).
1H NMR: (DMSO-d6): 4.65 (1H, m), 3.77 (2H, m), 3.51
(2H, m), 3.16 (1H, d, J = 9.6 Hz), 3.00 (1H, d,
J = 9.5 Hz), 2.51 (2H, m), 1.52 (2H, m).
13C NMR: (DMSO-d6): 176.1, 173.5, 82.0, 81.5, 56.7,
52.3, 51.8, 47.8, 32.8, 32.2, 30.5, 30.3.
13C NMR: (DMSO-d6): 172.2, 170.3, 141.7, 128.2, 128.1,
125.6, 78.6, 76.7, 53.0, 51.5, 38.9, 32.5, 30.7, 28.8, 28.6.
MP: 116–119 ꢁC.
MP: 135–137 ꢁC.
HRMS: C14H22N2O5 requires 299.1607; Found
299.1610.
HRMS (M+H) calcd 304.1549, found 304.1541.
4.10. 3-(2-Ethylphenylcarbamoyl)-7-oxabicyclo[2.2.1]hep-
tane-2-carboxylic acid (19)
4.6. 3-(4-Phenylbutylcarbamoyl)-7-oxabicyclo[2.2.1]
heptane-2-carboxylic acid (15)
1H NMR: (DMSO-d6): 11.85 (1H, COOH), 8.70 (1H,
NH), 7.65 (1H, d, J = 7.6Hz), 7.02–7.19 (3H, m), 4.85
(1H, d, J = 4.12Hz), 4.71 (1H, d, J = 4.32Hz), 3.07
(2H, q, J = 5.8Hz), 2.54 (2H, q, J = 7.5Hz), 1.53–1.64
(4H, m), 1.23 (3H, t, J = 7.6 Hz).
1H NMR: (DMSO-d6): 7.30 (1H, t), 7.15–7.29 (5H, m),
4.69 (1H, d), 4.43 (1H, d, J = 2.3Hz), 2.98 (2H, q,
J = 6.2Hz), 2.51(2H, quin, J = 7.3 Hz), 1.35–1.60
(8H, m).
13C NMR: (DMSO-d6): 172.7, 170.7, 142.1, 128.2, 125.6,
78.5, 77.0, 53.3, 52.3, 49.3, 38.2, 34.7, 28.5, 28.3.
13C NMR: (DMSO-d6): 172.0, 169.3, 135.7, 135.1, 128.2,
125.8, 124.4, 123.2, 79.0, 77.0, 54.1, 51.7, 28.4, 23.5, 14.0.
MP: 105–107 ꢁC.
MP: 171–172 ꢁC.
HRMS (M+H) calcd 318.1705, found 318.1712.
HRMS (M+H) calcd 290.1392, found 290.1388.

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S. G. Stewart et al. / Bioorg. Med. Chem. 15 (2007) 7301–7310
4.11. 3-(3-Ethylphenylcarbamoyl)-7-oxabicyclo[2.2.1]
heptane-2-carboxylic acid (20)
4.15. 3-(4-tert-Butylphenylcarbamoyl)-7-oxabicyclo[2.2.1]
heptane-2-carboxylic acid (24)
1H NMR (DMSO-d6): 11.1 (brs, 1H), 8.78 (1H, s, NH),
7.34 (1H, d, J = 7.7 Hz), 7.00 (1H, t, J = 7.7 Hz), 6.92
(1H, d, J = 7.2 Hz), 4.81 (1H, s), 4.69 (1H, d,
J = 4.2 Hz), 3.08 (1H, d, J = 9.7 Hz), 3.01 (1H, d,
J = 9.7 Hz), 2.22 (3H, s), 2.05 (3H, s), 1.52–1.59 (4H, m).
1H NMR (DMSO-d6): 11.89 (1H, brs), 9.48 (1H, s, 1H, NH),
7.42 (2H, d, J = 8.4 Hz), 7.26 (2H, d, J = 8.4 Hz), 4.77 (1H, s,
1H, CH), 4.61 (1H, d, J = 3.3 Hz), 3.03 (1H, d, J = 9.6 Hz),
2.91 (1H, d, J = 9.6 Hz), 1.45–1.60 (4H, m), 1.24 (9H, s).
13C NMR (DMSO-d6): 173.1, 169.9, 146.1, 137.5, 126.0
(2C), 119.8, 119.7, 54.4, 52.5, 34.8, 32.0 (3C), 29.8, 29.3.
13C NMR: (DMSO-d6): 173.2, 170.1, 137.4, 137.2, 129.9,
126.0, 125.9, 122.7, 79.9, 77.9, 54.7, 52.7, 29.6, 29.3,
21.1, 14.3.
MP: 179 ꢁC.
MP: 177 ꢁC.
HRMS (M+H) calcd 318.1705, found 318.1699.
HRMS (M+H) calcd 286.1079, found 286.1083.
4.16. 3-(2,4-Di-tert-Butylphenylcarbamoyl)-7-oxabicyclo
[2.2.1]heptane-2-carboxylic acid (25)
4.12. 3-(2,6-Dimethylphenylcarbamoyl)-7-oxabicyclo
[2.2.1]heptane-2-carboxylic acid (21)
1H NMR (DMSO-d6): 1.25 (18H, s), 1.49–1.62 (4H, m),
2.89 (2H, d, J = 9.5 Hz), 3.04 (2H, d, J = 9.5 Hz), 4.61
(1H, d, J = 4.0 Hz, 1H, CH), 4.84 (1H, s, 1H, CH),
7.05 (1H, s, 1H, CH–Ar), 7.39 (2H, s, 2H, 2· CH–Ar),
9.50 (1H, s, 1H, NH), 10.9 (1H, brs, 1H, COOH).
1H NMR: (DMSO-d6): 11.86 (1H, COOH), 8.96 (1H,
NH), 7.02 (3H, s), 4.78 (1H, d J = 3.6 Hz), 4.60 (1H, d
J = 4.7 Hz), 3.15 (1H, d J = 9.6 Hz), 2.90 (1H, d
J = 9.6 Hz), 2.13 (6H, s), 1.52–1.59 (4H, m).
13C NMR (DMSO-d6): 173.2, 169.9, 151.4, 139.6, 117.5,
114.3, 79.8, 77.8, 54.6, 52.3, 35.3, 32.1 (6C), 29.8, 29.4.
13C NMR: (DMSO-d6): 172.2, 168.8, 135.2, 135.1, 127.4,
126.1, 79.3, 76.6, 52.9, 51.0, 29.0, 28.5, 18.1.
MP: 182 ꢁC.
MP: 193–195 ꢁC.
HRMS (M+H) calcd 374.2331, found 374.2335.
HRMS (M+H) calcd 290.1392, found 290.1396.
4.17. 3-(Naphthalen-2-ylcarbamoyl)-7-oxabicyclo[2.2.1]
heptane-2-carboxylic acid (26)
4.13. 3-(2,3-Dimethylphenylcarbamoyl)-7-oxabicyclo
[2.2.1]heptane-2-carboxylic acid (22)
1H NMR (DMSO-d6): 10.95 (1H, brs), 9.82 (1H, s),
7.74–7.83 (3H, s), 7.36–7.53 (3H, m), 4.79 (1H, s), 4.67
(1H, s), 3.17 (2H, d, J = 9.9 Hz), 2.96 (2H, d,
J = 9.9 Hz), 1.51–1.62 (4H, m).
1H NMR: (DMSO-d6): 10.9 (1H, brs), 9.75 (1H, s, NH),
7.73 (1H, s), 7.43 (1H, d, J = 8.1 Hz), 7.27 (1H, t,
J = 7.8 Hz), 7.11 (1H, d, J = 7.3 Hz), 4.76 (1H, s), 4.63
(1H, d, J = 3.8), 4.10 (1H, s), 3.03 (2H, d, J = 9.6 Hz),
2.93 (2H, d, J = 9.6 Hz), 1.52–1.54 (4H, m).
13C NMR (DMSO-d6): 173.1, 170.5, 137.7, 134.3, 130.5,
129.0, 128.3, 128.0, 127.2, 125.3, 120.9, 116.0, 79.6, 77.8,
54.5, 52.7, 29.8, 29.3.
13C NMR: (DMSO-d6): 171.9, 169.4, 139.3, 128.8, 126.1,
122.3, 121.8, 119.9, 83.3, 79.4, 78.4, 77.0, 53.5, 51.8,
28.8, 28.3.
MP: 237–238 ꢁC.
MP: 166 ꢁC.
HRMS (M+H) calcd 312.1236, found 312.1242.
4.18. 3-(4-Hexyloxyphenylcarbamoyl)-7-oxabicyclo[2.2.1]
heptane-2-carboxylic acid (27)
HRMS (M+H) calcd 290.1392, found 290.1400.
4.14. 3-(2,4,6-Trimethylphenylcarbamoyl)-7-oxabicy-
clo[2.2.1]heptane-2-carboxylic acid (23)
1H NMR (DMSO-d6): 10.85 (1H, brs), 9.41 (1H, s), 7.39 (2H,
d, J = 8.9 Hz), 6.82 (2H, d, J = 8.9 Hz), 4.84 (1H, d, J = 3.8
Hz), 4.61 (1H, d, J = 3.8 Hz), 3.89 (2H, t, J = 6.5 Hz), 3.01
(1H, d, J = 9.6 Hz), 2.91 (1H, d, J = 9.6 Hz), 1.51–1.67
(6H, m), 1.34–1.36 (4H, m), 0.88 (3H, t, J = 6.9 Hz).
1H NMR (DMSO-d6): 11.85 (1H, brs), 8.87 (1H, s, 1H,
NH), 6.82 (2H, s), 4.77 (1H, s), 4.58 (1H, d, J = 4.5 Hz),
3.12 (1H, d, J = 9.4 Hz), 2.88 (1H, d, J = 9.4 Hz), 2.19
(3H, s), 2.08 (6H, s), 1.51–1.66 (4H, m).
13C NMR (DMSO-d6): 173.1, 169.7, 155.4, 133.2, 121.7
(2C), 115.2 (2C), 79.6, 77.7, 68.5, 54.3, 52.5, 29.8, 29.3
(2C), 28.6, 22.8, 14.8.
13C NMR (DMSO-d6): 172.2, 168.8, 135.0, 134.9, 132.4,
128.0, 79.3, 76.6, 52.9, 50.9, 29.0, 28.5, 18.0, 17.9 (2C).
MP: 156 ꢁC.
MP: 187 ꢁC.
HRMS (M+H) calcd 362.1968, found 362.1965.
HRMS (M+H) calcd 304.1549, found 304.1553.

S. G. Stewart et al. / Bioorg. Med. Chem. 15 (2007) 7301–7310
7309
4.19. 3-(4-Methoxyphenylcarbamoyl)-7-oxabicyclo[2.2.1]
heptane-2-carboxylic acid (28)
4.23. 3-(3-Hydroxymethylphenylcarbamoyl)-7-oxabicy-
clo[2.2.1]heptane-2-carboxylic acid (32)
1H NMR (DMSO-d6): 9.39 (1H, s, NH), 7.40 (2H, d,
J = 8.9 Hz), 6.83 (2H, d, J = 8.9 Hz), 4.76 (1H, d,
J = 3.7 Hz), 4.60 (1H, d, J = 3.7 Hz), 3.69 (3H, s), 3.09 (1H,
d, J = 9.6 Hz), 2.90 (1H, d, J = 9.6 Hz), 1.51–1.56 (4H, m).
1H NMR: (DMSO-d6): 10.9 (1H, brs), 9.54 (1H, s, NH),
7.49 (1H, s), 7.37 (1H, d, J = 8.0 Hz), 7.19 (1H, t,
J = 7.7 Hz), 7.76 (1H, d, J = 7.4 Hz), 5.11 (1H, brs),
4.75 (1H, d, J = 3.8 Hz), 4.62 (1H, d, J = 3.8 Hz), 4.44
(2H, s), 3.05 (1H, d, J = 9.6 Hz), 2.92 (1H, d,
J = 9.6 Hz), 1.49–1.60 (4H, m).
13C NMR (DMSO-d6): 173.1, 169.7, 156.0, 133.3, 121.7
(2C), 114.6 (2C), 79.6, 77.8, 56.1, 54.4, 52.5, 29.8, 29.3.
13C NMR: (DMSO-d6): 173.1, 170.1, 143.9, 140.0, 129.1,
121.9, 118.4, 118.2, 79.6, 77.8, 63.7, 54.5, 52.5, 29.8,
29.3.
MP: 159 ꢁC.
HRMS (M+H) calcd 292.1185, found 292.1188.
MP: 132 ꢁC.
4.20. 3-(4-Methylsulfanylphenylcarbamoyl)-7-oxabicyclo
[2.2.1]heptane-2-carboxylic acid (29)
HRMS (M+H) calcd 292.1185, found 292.1183.
1H NMR (DMSO-d6):11.9(1H,s),9.61(1H,s,NH),7.47(2H,
d, J = 8.4 Hz), 7.19 (2H, d, J = 8.4 Hz), 4.84 (1H, d, J = 3.9
Hz), 4.62 (1H, d, J = 3.9 Hz), 3.03 (1H, d, J = 9.6 Hz), 2.92
(1H, d, J = 9.6 Hz), 2.42 (3H, s), 1.48–1.60 (4H, m).
4.24. 3-(3,4-Difluorophenylcarbamoyl)-7-oxabicyclo[2.2.1]
heptane-2-carboxylic acid (33)
1H NMR: (DMSO-d6): 9.88 (1H, s, NH), 7.69 (1H, m),
7.30 (1H, m), 7.19 (1H, m), 4.76 (1H, br), 4.63 (1H, br),
2.97 (2H, m), 1.67–1.47 (4H, m).
13C NMR (DMSO-d6): 173.0, 170.1, 137.8, 132.2, 128.1
(2C), 120.8 (2C), 79.5, 77.7, 54.3, 52.6, 29.8, 29.3, 16.7.
13C NMR: (DMSO-d6): 172.0, 169.6, 150.5, 147.3, 143.5,
136.1, 117.2, 108.0, 78.4, 76.9, 53.2, 51.8, 28.8, 28.3.
MP: 172–173 ꢁC.
HRMS (M+H) calcd 308.0957, found 308.0962.
MP: 174–176 ꢁC.
4.21. 3-(2-Methylsulfanylphenylcarbamoyl)-7-oxabicyclo
[2.2.1]heptane-2-carboxylic acid (30)
HRMS (M+H) calcd 298.0891, found 298.0893.
1H NMR (DMSO-d6): 1.57 (m, 4H, 2· CH2), 2.37
(s, 3H, CH3), 3.09 (s, 2H, 2· CH), 4.75 (d, J = 4.2 Hz,
1H, CH), 4.88 (s, 1H, CH), 7.08 (t, J = 7.5 Hz, 2H,
2· CH–Ar), 7.19 (t, J = 7.5 Hz, 1H, CH–Ar), 7.38 (d,
J = 7.8 Hz, 1H, CH–Ar), 7.84 (d, J = 7.8 Hz, 1H, CH–
Ar), 8.98 (s, 1H, NH), 12.2 (s, 1H, COOH).
4.25. 3-(4-Trifluoromethylphenylcarbamoyl)-7-oxabicy-
clo[2.2.1]heptane-2-carboxylic acid (34)
1H NMR: (DMSO-d6): 11.97 (1H, brs), 10.01 (1H, s,
NH), 7.73 (2H, d, J = 8.4 Hz), 7.61 (2H, d,
J = 8.4 Hz), 4.77 (1H, br), 4.66 (1H, br), 3.07 (1H,
d, J = 9.6 Hz), 2.96 (1H, d, J = 9.6 Hz), 1.58–1.50
(4H, m).
13C NMR (DMSO-d6): 16.5 (CH3), 28.3 (CH2), 28.4 (CH2),
51.8 (CH), 54.6 (CH), 77.0 (CH), 78.9 (CH), 122.2 (C–Ar),
124.5 (CH–Ar), 126.6 (CH–Ar), 128.2 (C–Ar), 129.7 (CH–
Ar), 137.1 (CH–Ar), 169.6 (CON), 172.0 (COOH).
13C NMR: (DMSO-d6): 172.0, 169.8, 142.7, 126.1–123.5
(d, J = 894.0 Hz), 125.8 (q, J = 15.0 Hz), 123.5, 123.1,
122.7, 122.5, 122.3 (sextet, J = 60 Hz), 78.4, 76.9, 53.4,
51.8, 28.8, 28.3.
MP: 135 ꢁC.
MP: 189–191 ꢁC.
HRMS (M+H) calcd 308.0957, found 308.0952.
4.22. 3-(2-Hydroxymethylphenylcarbamoyl)-7-oxabicyclo
[2.2.1]heptane-2-carboxylic acid (31)
HRMS (M+H) calcd 330.0953, found 330.0955.
4.26. 3-(4-Morpholin-4-ylphenylcarbamoyl)-7-oxabicy-
clo[2.2.1]heptane-2-carboxylic acid (35)
1H NMR (DMSO-d6): 8.99 (1H, s, NH), 7.63 (1H, d,
J = 7.9 Hz), 7.32 (1H, d, J = 7.4 Hz), 7.19 (1H, t,
J = 7.1 Hz), 7.06 (1H, t, J = 7.1 Hz), 5.22 (1H, brs),
4.69 (1H, d, J = 4.2 Hz), 4.45 (2H, s), 4.08 (1H, s),
3.03 (2H, t, J = 10.3 Hz), 1.51–1.62 (4H, m, 4H).
1H NMR (DMSO-d6): 10.9 (1H, brs), 9.35 (s, 1H, NH),
7.36 (1H, d, J = 8.9 Hz), 6.85 (1H, d, J = 8.9 Hz), 4.76
(1H, d, J = 2.1 Hz), 4.60 (1H, d, J = 3.7 Hz), 2.88–3.02
(6H, m, 6H), 1.47–1.56 (4H, m).
13C NMR (DMSO-d6): 172.7, 169.9, 136.4, 133.9, 127.9,
127.4, 124.4, 123.5, 79.3, 77.6, 60.7, 54.7, 52.4, 29.1, 29.0.
13C NMR (DMSO-d6): 173.1, 169.6, 148.0, 132.5, 121.2
(2C), 116.3 (2C), 79.6, 77.7, 67.0, 54.4, 52.5, 50.0, 29.8,
29.3.
MP: 145–146 ꢁC.
HRMS (M+H) calcd 292.1185, found 292.1190.
HRMS (M+H) calcd 347.1607, found 347.1611.

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S. G. Stewart et al. / Bioorg. Med. Chem. 15 (2007) 7301–7310
20. Chae, W. J.; Choi, J. M.; Yang, J. J.; Lee, S. K. Yonsei
Med. J. 2004, 45, 978–990.
Acknowledgments
21. Ubukata, M.; Cheng, X. C.; Isono, K. J. Chem. Soc.
Chem. Commun. 1990, 244–246.
22. Mitsuhashi, S.; Shima, H.; Tanauma, N.; Matsuura, N.;
Takekawa, M.; Urano, T.; Kataoka, T.; Ubukata, M.;
Kikuch, K. J. Biol. Chem. 2003, 278, 82–88.
The work received financial support from the Hunter
Medical Research Institute and the Biotechnology Inno-
vation Fund, AusIndustry, Australia.
23. Lee, J.-H.; Lee, J.-S.; Kim, S. E.; Moon, B.-S.; Kim, Y.-C.;
Lee, S.-K.; Lee, S.-K.; Choi, K.-Y. Mol. Cancer Ther.
2006, 5, 3222–3231.
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