Norcantharimides, synthesis and anticancer activity: Synthesis of new norcantharidin analogues and their anticancer evaluation

Article abstract of DOI:10.1016/j.bmc.2007.06.034

A range of amines was reacted with norcantharidin (2) to provide the corresponding norcantharimides (9-43). Treatment of norcantharidin with allylamine afforded the corresponding allyl-norcantharimide (20) which was amenable to epoxidation (mCPBA, 22) and

Full text of DOI:10.1016/j.bmc.2007.06.034

Bioorganic & Medicinal Chemistry 15 (2007) 6126–6134
Norcantharimides, synthesis and anticancer activity: Synthesis
of new norcantharidin analogues and their anticancer evaluation
Timothy A. Hill,a Scott G. Stewart,a Stephen P. Ackland,b Jayne Gilbert,b
Benjamin Sauer,a Jennette A. Sakoffb and Adam McCluskeya,*
aChemistry, Chemistry Building, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia
bDepartment of Medical Oncology, Newcastle Mater Misericordiae Hospital, Waratah, NSW 2298, Australia
Received 20 March 2007; revised 12 June 2007; accepted 13 June 2007
Available online 20 June 2007
Abstract—A range of amines was reacted with norcantharidin (2) to provide the corresponding norcantharimides (9–43). Treatment
of norcantharidin with allylamine afforded the corresponding allyl-norcantharimide (20) which was amenable to epoxidation
(mCPBA, 22) and subsequent ring opening (MeOH/H+; 23) or alternatively, osmylation (OsO4/NMO; 24). These simple synthetic
modifications of 2 facilitated the development of a novel series of norcantharimides displaying modest to good broad spectrum cyto-
toxicity against HT29 and SW480 (colorectal carcinoma); MCF-7 (breast adenocarcinoma); A2780 (ovarian carcinoma); H460 (lung
carcinoma); A431 (epidermoid carcinoma); DU145 (prostate carcinoma); BE2-C (neuroblastoma); and SJ-G2 (glioblastoma). Ana-
logues possessing a C10, C12 or C14 alkyl chain or a C12 linked bis-norcantharimide displayed the highest levels of cytotoxicity.
Crown copyright ꢀ 2007 Published by Elsevier Ltd. All rights reserved.
1. Introduction
siderable pressure to develop new treatments and new
therapeutic approaches to the treatment of cancer.
More than five decades of research effort in cancer drug
discovery and development have provided less than 100
approved products for the treatment of malignancy.1
Although major advances have been made in the chemo-
therapeutic management of some patients, particularly
in haematologic malignancies, one-half of all cancer pa-
tients either do not respond to therapy, relapse follow-
ing initial response or ultimately die from their
metastatic disease. For such patients a better systemic
therapy offers the only chance for prolonged survival
or ultimately a cure. Many mechanisms of tumour cell
resistance to conventional agents involve alterations in
DNA cell cycling events. New agents that specifically
target cell cycle events have the capacity to overcome
resistance to chemotherapy, or possibly induce tumour
cell death when used alone. This requires continued re-
search to discover novel therapeutic products that can
be used in combination with biologic agents and im-
mune therapies to eradicate systemic disease not curable
by surgery or irradiation. There is, understandably, con-
Over the past decade we have been exploring the con-
nection between protein phosphatase inhibition (PP1
and PP2A) and anticancer activity.2–6 In particular we
have focused on the synthetic modification of canthari-
din (1) and norcantharidin (2) (Chart 1). Clinical evalu-
ations of 1 have indicated promise in the treatment of
liver tumours and the KB cell line at low concentra-
tions.7–10 A direct outcome of our early work in this area
was the discovery that various norcantharimides dis-
played modest anticancer activity and as such they have
proved to be interesting lead compounds in the search
for new anticancer agents. Others, notably Lin et al.,
have also reported the synthesis and anticancer activity
of the cantharimides, and in doing so reduced toxicity
whilst maintaining biological activity.11,12 However, we
felt that given the known nephrotoxicity of cantharidin,
O
O
O
O
O
O
Keywords: Cantharidin; Norcantharidin; Norcantharimides; Cytotox-
icity; Anticancer.
O
O
1
2
*
Corresponding author. Tel.: +61 249 216486; fax: +61 249 215472;
e-mail: Adam.McCluskey@newcastle.edu.au
Chart 1.
0968-0896/$ - see front matter Crown copyright ꢀ 2007 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.06.034

T. A. Hill et al. / Bioorg. Med. Chem. 15 (2007) 6126–6134
6127
1, and the relative ease of synthesis associated with prep-
aration of large quantities of norcantharidin, 2, that the
norcantharimides also presented an ideal opportunity to
develop a library of potential anticancer agents.3,6
As can be seen both 1 and 2 are potent, broad spectrum
anticancer agents with 1 being typically 10-fold more
potent than 2. With the alkyl-substituted norcanthari-
mides 9–19, modest to poor activity is observed. The
only instances of noteworthy cytotoxicity are observed
with 16, 17 and 18, all of which possess a long alkyl
chain (C10, C12 and C14, respectively). Presumably these
alkyl chains assist in penetration of the cell membrane
making these analogues bio-available. Shorter (9–15),
longer (18), branched or cyclic alkyl groups display no
activity. Notwithstanding this, the data obtained suggest
that 16–18 have potential as anticancer agents. The
introduction of a terminal double bond, 20 and 21,
has essentially no impact on activity (but does allow
easy functionalisation—see Scheme 2), although the al-
lyl substitution is more active than the butenyl analogue.
This differential reactivity is most likely an artefact of
the greater stability/reactivity of the allyl group with
the potential delocalisation of any charge back to the
norcantharimide nitrogen. This stabilisation is not pos-
sible with the corresponding butenyl substituent. It is
also of note that the introduction of the terminal double
bond returns a modest increase in activity relative to the
parent propyl analogue 10. Both 10 and 20 show very
modest levels of activity against SW480 (10, 45%; 20,
50% at 100 lM, respectively) and MCF-7 (20,
GI50 = 80 12 lM) cells lines, promising but of no great
worth at this stage.
2. Results and discussion
The key starting material in this work was the readily
synthesisable 5,6-dehydronorcantharidin, 5, which was
prepared on a large scale through exo-selective cycload-
dition of the relatively cheap furan, 3, and maleic anhy-
dride. Subsequent hydrogenation of 5 (H2, 40 psi, 10%
Pd–C) following a modified procedure of Eggelte et al.
provided the starting norcantharidin (2) in excellent
yields.13
Treatment of 2 with an appropriate primary amine and
NEt3 facilitated a condensation reaction to provide a
series of cyclic imides (6, Scheme 1, compounds 9–43,
Tables 1 and 2) similar to our procedure for the synthe-
sis of related amino acid substituted N-derivatives.3
Our approaches to medicinal chemistry have relied on
the rapid and robust generation of small-targeted com-
pound libraries for bioassay.3–6 Thus, simple reflux
afforded the desired analogues in good to excellent yields
(30–90%, Tables 1 and 2), contrasting the requirement
for high pressure approaches with the cantharimide ana-
logues reported by Lin et al.11 Reaction yields were
essentially as anticipated with variations reflecting the
nucleophilicity of the parent amine.
Suitably encouraged by that simple norcantharimides
displayed reasonable cytotoxicity, we next examined
the introduction of higher levels of functionality. In this
instance we limited our explorations to terminally
substituted analogues. Commencing with the easily
functionalised allyl unit of 20, mCPBA mediated epoxi-
dation gave 22, which underwent a subsequent (1S)-(+)-
10-camphorsulfonic acid mediated methanolysis to the
methoxy alcohol 23. Alternatively osmylation with
OsO4 in the presence of NMO afforded the diol 24
(Scheme 2). These modification of the allyl substituent
gave rise to the most interesting structure activity data
thus far. Cytotoxicity screening showed the parent epox-
ide 22 possessed very low levels of cytotoxicity (ca. 25%
at 100 lM across all cell lines evaluated). The methoxy
alcohol 23 displays lower levels of cytotoxicity than
epoxide 22. Notwithstanding this introduction of a diol
unit (24) gave an analogue displaying considerably high-
er levels of cytotoxicity, than 22, with GI50 values deter-
mined for four cell lines (SW480, MCF-7, A2780 and
BE2-C with GI50’s of 62 2, 46 4, 59 2 and
70 4 lM, respectively) and also modest levels of activ-
ity at the remaining five cell lines (ca. 45% at 100 lM).
Removal of the secondary alcohol (24 and 25) results
in a significant reduction of cytotoxicity (ca. 20% at
100 lM across all cell lines evaluated), strongly suggest-
ing that both –OH groups are required for cytotoxicity.
Indeed the requirement for two free –OH groups is con-
firmed by the lack of activity associated with analogues
25–29. Encouraged by the increase in potency observed
via the introduction of oxygen bearing functionallity we
next sought to introduce such functionallity via a termi-
nal carboxylate. Disappointingly these carboxylate ana-
logues, 30 and 31, failed to elicit any noteworthy levels
We first examined a series of simple alkyl norcanthari-
mide analogues, developed based on linear alkyl amines
(Table 1). The data arising from subsequent cell survival
screening against a panel of nine human cancer cell lines
routinely grown in our laboratory: HT29 and SW480
(colorectal carcinoma); MCF-7 (breast adenocarci-
noma); A2780 (ovarian carcinoma); H460 (lung carci-
noma); A431 (epidermoid carcinoma); DU145
(prostate carcinoma); BE2-C (neuroblastoma); and SJ-
G2 (glioblastoma) are shown in Table 1. Note that can-
tharidin (1) and norcantharidin (2) are regularly run in
our laboratories as comparative controls.
O
O
O
a
b
O
O
O
O
O
+
O
O
O
O
O
3
4
5
2
c
O
N
R
O
6
Scheme 1. Reagents and conditions: (a) rt, 48 h, ether; (b) 4 atm H2,
10% Pd–C, acetone, 3-days; (c) RNH2, PhCH3 reflux, 36 h.

6128
T. A. Hill et al. / Bioorg. Med. Chem. 15 (2007) 6126–6134
Table 1. Cytotoxicity of cantharidin (1), norcantharidin (2) and norcantharimides (9–21) in a panel of human cancer cell lines
Compoundb
R
Tumour cell linea
HT29
Colon
SW480
Colon
MCF-7
Breast
A2780
Ovarian
H460
Lung
A431
Skin
DU145
Prostate
BE2-C SJ-G2
Neuronal Brain
O
O
N
R
O
1
2
3.2 0.1
5
4.5 0.3
6
7.5 0.4
4
4.4 0.3
1
3.3 0.2
3
2.9 0.2
31
2.1 0.3
3
3.7 0.6
6
1.7 0.1
3
57
44
68
38
45
1
28
43
23
9
10
11
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
16 11
45 44
<10
<10
()
()
2
12
<10
<10
<10
<10
<10
<10
<10
<10
<10
13
14
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
4
15
16
17
18
19
20
70
20
5
1
4
0
65
59
4
0
2
4
88 18
50 10
<10
>100
53
61
52
3
5
4
3
44 11
68 16
54 11
()
()
()
()
6
44
52
4
8
7
44
40
7
4
4
83
66
4
4
2
39
40
4
6
4
72
58
2
2
5
8
25
55
4
35
10
12
16
19
52
43
35
66
7
47
73
50
49
<10
19
<10
<10
<10
<10
20
<10
32
<10
<10
62
<10
2
()
9
50 26
96 14
80 12
67 12
8
5
15 17
5
29
21
<10
<10
<10
<10
<10
<10
<10
<10
<10
Cytotoxicity levels are first expressed as % inhibition at 100 lM drug concentration (in italics) and if potent, as Growth Inhibition, GI50, lM.
a Data were calculated after 72 h of continuous drug exposure, values are means ( SEM) of 3–6 experiments.
b Compound, compound number.
O
O
O
a
b
O
O
OH
O
N
N
N
O
OCH
3
O
O
O
20
22
23
c
O
O
OH
N
OH
O
24
Scheme 2. Reagents and conditions: (a) mCPBA, rt 16 h; (b) (1S)-(+)-10-camphorsulfonic acid/MeOH; (c) OsO4/NMO, acetone/H2O, 80 ꢁC 16 h.

T. A. Hill et al. / Bioorg. Med. Chem. 15 (2007) 6126–6134
6129
Table 2. Cytotoxicity of norcantharimides (22–43) in a panel of human cancer cell lines
Compoundb
R
Tumour cell linea
HT29
Colon
SW480
MCF-7
Breast
A2780
Ovarian
H460
Lung
A431
Skin
DU145
Prostate
BE2-C SJ-G2
Neuronal Brain
O
O
N
R
O
O
22
23
24
25
12
3
4
30
6
10 16
40
6
17
5
40
9
22
5
20 13
25
4
5
O
<10
33
3
4
<10
<10
<10
<10
<10
<10
OH
OH
OH
39 12
>100
69 29 >100
62
>100
59
32 11
>100
60 19
>100
67 24
>100
73
7
4
27
2
46
2
70
>100
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
31 12
18
16
3
2
45 22
51 13
<10
15
18
2
2
16
3
1
<10
6
<10
<10
<10
<10
<10
<10
12
<10
22
<10
12
OH
21
<10
<10
<10
<10
14
4
14
<10
<10
<10
<10
12
3
9
5
OH
5
<10
<10
<10
<10
11
<10
<10
<10
<10
<10
<10
<10
<10
11
<10
<10
<10
<10
<10
<10
<10
<10
<10
3
OH
OH
OH
OH
<10
<10
O
<10
()
3
O
7
5
1
1
47 35
52 29
2
0
7
5
6
5
22 21
()
5
OH
29
29
12
4
7
2
18
30
3
39
<10
<10
54
8
2
N
O
<10
<10
63
<10
<10
<10
<10
<10
<10
<10
<10
50
<10
<10
25
<10
<10
24
<10
<10
42
N
O
N
O
4
8
4
78 24 >100
90 11
3
0
4
3
1
9
6
5
6
4
7
47
21
3
22
4
15
3
12
1
0
27
<10
13
14
44
OH
NO
60
44 11 >100
53 11
37
13
45
23
2
<10
45
<10
24
<10
<10
<10
23
<10
14
<10
<10
12
<10
29
<10
11
CO H
2
4
8
4
9
4
64 10
71 22
41 26
20
1
6
5
2
5
1
3
O
O
45
29
29 11
31 15
<10
<10
6
11
<10
<10
O
33 10
13
18
5
21
7
<10
13
H N
2
(continued on next page)

6130
T. A. Hill et al. / Bioorg. Med. Chem. 15 (2007) 6126–6134
Table 2 (continued)
Compoundb
R
Tumour cell linea
HT29
Colon
SW480 MCF-7 A2780 H460
Breast Ovarian Lung
A431
Skin
DU145
Prostate Neuronal Brain
BE2-C
SJ-G2
O
O
N
R
O
O
O
42
43
O
O
O
O
10
7
16
5
52 41 31 26
18
7
10
5
13
5
10
8
10
8
N
N
O
O
O
O
8.3 0.7 24
4
18
0
19
1
31
7
18
4
60
6
17
4
43 10
N
()
N
10
O
O
Cytotoxicity levels are first expressed as % inhibition at 100 lM drug concentration (in italics) and if potent, as Growth Inhibition, GI50, lM.
a Data were calculated after 72 h of continuous drug exposure, values are means ( SEM) of 3–6 experiments.
b Compound, compound number.
of cytotoxicity. However the hexanoic acid substituted
31 does show improved activity against MCF-7 and
A2780 cell lines when compared with the analogous hex-
yl alcohol 26 and hexyl substituted 13 suggesting that
the introduction of a terminal acid moiety is beneficial
to cytotoxicity. The introduction of a morpholine sub-
stituent, 32–34, had no effect on activity, which is con-
tray to our findings with the corresponding ring
opened analogues.14
broad spectrum cytotoxicity in excess of that observed
for norcantharidin (2) with GI50 values ranging from
8.3 0.7 to 60 6 lM across the panel of cell lines
examined.
3. Conclusions
Simple modification of the parent norcantharidin (2) has
allowed the development of a new series of norcanthari-
mides with modest to good cytotoxicities. The most po-
tent analogues synthesised contained either a C10, C12,
C14 alkyl chain (analogues 16–18), a 1,2-diol moiety
(23) or a dodecyl-linked second norcantharimide moiety
(43). Of the analogues generated the dodecyl-linked bis-
norcantharimide (43) was the most potent analogue dis-
playing lM potent cytotoxicities against all the cell lines
examined at levels that improve on the lead norcantha-
ridin (2).
Given our lack of improvement via terminally substi-
tuted linear or branched chains and the reported activity
of the corresponding aromatic substituted canthari-
mides by Lin,11,12 we synthesised a range of the equiva-
lent phenyl substituted norcantharimides (Table 2,
35–38).3 In all instances we observed no noteworthy
cytotoxicity at 100 lM drug dose (our primary screen
prior to GI50 determination). Interestingly our most
active aniline derived analogues, 35 and 37, correspond
well with the only active cantharimide aniline analogue
reported by Lin.11,12 However, Lin’s cantharidin
analogue is significantly more potent against the cell
lines examined (HL-60 IC50 = 8.4 lM; and Hep G2
IC50 = 69 lM) a pattern which is also seen when com-
paring the parent cantharidin (1) and norcantharidin
(2). In our hands no beneficial effect was observed by
generation of a small series of benzyl substituted ana-
logues (39–41), with no appreciable activity noted in
all cases. As we were unable to increase the potency
of our norcantharimides via the introduction of an
aromatic ring, we directed our synthetic efforts towards
linear, norcantharimide dimers of the type recently
reported by Noda et al.15
4. Experimental
4.1. Materials and methods
All reagents were of commercial quality and were used
as received (Aldrich). Solvents were dried and purified
using standard techniques. Reactions were monitored
by TLC, on aluminium plates coated with silica gel with
fluorescent indicator (Merck 60 F254).
Unless otherwise noted, NMR spectra were recorded in
CDCl3 at 300 MHz for 1H and at 75 MHz for 13C (Bru-
ker Advance 300MX). GCMS was performed using a
Shimadzu GCMS-QP2010. The instrument uses a quad-
rupole mass spectrometer and detects samples via elec-
tron impact ionisation (EI) or chemical ionisation
using methane (CI). The University of Wollongong Bio-
molecular Mass Spectrometry Laboratory analysed
samples for HRMS. The spectra were run on the VG
Treatment of 1,3-diaminopropane and 1,12-diaminodo-
decane with 0.5 equiv of 2 under standard conditions
afforded bis-norcantharimides 42 and 43. Bis-norcant-
harimide 43 with a dodecyl linker proved to be the most
cytotoxic of all the analogues generated herein with

T. A. Hill et al. / Bioorg. Med. Chem. 15 (2007) 6126–6134
6131
Autospec-oa-tof tandem high resolution mass spectrom-
eter using CI +ve (Chemical Ionisation), with methane
as the carrier gas and PFK as the reference.
10.5, 20.3, 28.0 (2C), 40.0, 49.3, 78.4, 176.6 (2C). HR-
MS m/z: (calcd for C11H15NO3: 209.10519).
4.1.6. 4-Butyl-10-oxa-4-azatricyclo[5.2.1]decane-3,5-dione
1
4.1.1. Cell culture and stock solutions. Stock solutions
were prepared as follows and stored at À20 ꢁC: Can-
tharidin (Biomol, USA) as a 30 mM solution in dimeth-
(11). Isolated as a white solid, 93%, mp 82–83 ꢁC. H
NMR (CDCl3): d 0.90 (3H, t, J = 7.2 Hz), 1.28 (2H,
sep, J = 7.8 Hz), 1.49–1.59 (4H, m), 1.82–1.85 (2H, m),
2.83 (2H, s), 3.45 (2H, t, J = 7.3 Hz), 4.85 (2H, q,
J = 0.7 Hz). 13C NMR (CDCl3): d 12.9, 19.3, 28.0 (2C),
29.0, 38.2, 49.3 (2C) 78.4, 176.6 (2C). HR-MS m/z: (calcd
for C12H17NO3: 223.12084).
ylsulfoxide (DMSO); norcantharidin as
a 30 mM
solution in water and norcantharidin analogues as
40 mM solutions in DMSO. All cell lines were cultured
at 37 ꢁC, under 5% CO2 in air, and were maintained in
Dulbecco’s modified Eagle’s medium (Trace Biosciences,
Australia) supplemented with 10% foetal bovine serum,
10 mM sodium bicarbonate penicillin (100 IU/ml), strep-
tomycin (100 lg/ml) and glutamine (4 mM).
4.1.7. 4-Sec-butyl-10-oxa-4-azatricyclo[5.2.1]decane-3,5-
dione (12). Isolated as a white solid, 98%, mp: 44–46 ꢁC.
1H NMR (DMSO-d6): 0.79 (3H, t, J = 7.5 Hz), 1.30
(3H, d, J = 7.0 Hz), 1.64–1.52 (2H, m), 1.80–1.92 (4H,
m), 2.77 (2H, m), 4.03 (1H, m), 4.82 (2H, s). 13C NMR
(DMSO-d6): 10.9, 17.5, 25.9, 28.6, 49.5, 50.1, 79.2,
177.5. HR-MS m/z: (calcd for C12H17NO3: 223.12084).
4.1.2. In vitro growth inhibition assay. Cells in logarith-
mic growth were transferred to 96-well plates. Cytotox-
icity was determined by plating cells in duplicate in
100 mL medium at a density of 2500–4000 cells/well.
On day 0 (24 h after plating) when the cells were in log-
arithmic growth, 100 lL medium with or without the
test agent was added to each well. After 72-h drug expo-
sure growth inhibitory effects were evaluated using the
MTT (3-[4,5-dimethyltriazol-2-yl]-2,5-diphenyl-tetrazo-
lium bromide) assay and absorbance read at 540 nm.
Percentage growth inhibition was determined at a fixed
drug concentration of 100 lM. A value of 100% is indic-
ative of total cell growth inhibition. Those analogues
showing appreciable percentage growth inhibition
underwent further dose–response analysis allowing for
the calculation of a GI50 value. This value is the drug
concentration at which cell growth is 50% inhibited
based on the difference between the optical density val-
ues on day 0 and those at the end of drug exposure.2
4.1.8. 4-Hexyl-10-oxa-4-azatricyclo[5.2.1]decane-3,5-dione
(13). Isolated as a colourless oil, 87%. 1H NMR (CDCl3):
d 0.85 (4H, m), 1.25 (8H, m), 1.57 (4H, m), 1.84 (2H, m),
2.84 (2H, s), 3.43 (2H, t, J = 7.1 Hz), 4.85 (2H, q,
J = 2.4 Hz). 13C NMR (CDCl3): d 13.4, 21.9, 25.7, 26.9,
28.0, 30.7, 38.5, 49.3, 78.5, 176.7. HR-MS m/z: (calcd
for C14H21NO3: 251.15214).
4.1.9. 4-Cyclohexyl-10-oxa-4-azatricyclo[5.2.1]decane-3,5-
dione (14). Isolated as a white solid, 82%, mp 97–99 ꢁC.
1H NMR (DMSO-d6): 1.16 (4H, m), 1.53 (6H, m), 1.77
(4H, m), 2.05 (2H, m), 2.73 (2H, s), 3.86 (1H, m), 4.78
(2H, m). 13C NMR (DMSO-d6): 25.0, 25.8, 28.5, 28.6,
49.4, 51.9, 79.1, 177.3. HR-MS m/z: (calcd for
C14H19NO3: 249.13649).
4.1.3. Chemistry. General synthetic procedure for the
synthesis of norcantharimides: Amine (1 equiv,
2.97 mmol) was added to a magnetically stirred solution
of norcantharidin 2 (1.0 g, 2.97 mmol) and triethylamine
(1.5 mL) in toluene (15 mL). This solution was refluxed
for 36 h before being cooled, diluted with EtOAc
(45 mL), washed with NaHCO3 (2· 5 mL, saturated
solution), dried (MgSO4), filtered and concentrated un-
der reduced pressure. The resulting crude norcanthari-
mide was either recrystallised from EtOAc/Hexane or
subjected to flash chromatography (ꢀ40% EtOAc/Hex-
ane) to afford norcantharimides 9–35 (ꢀ7–98% yield,
depending on amine).
4.1.10. 4-Octyl-10-oxa-4-azatricyclo[5.2.1]decane-3,5-dione
(15). Isolated as a white solid, 72%, mp 36–37 ꢁC. H
1
NMR (CDCl3): d 0.85 (3H, t, J = 6.9 Hz), 1.24 (10H, s),
1.52–1.59 (4H, m), 1.81–1.84 (2H, m), 2.82 (2H, s), 3.43
(2H, t, J = 7.5 Hz), 4.84 (2H, q, J = 1.0 Hz). 13C NMR
(CDCl3): d 13.4 , 22.0, 21.9, 26.1, 27.0, 28.0 (2C), 28.4,
28.5, 31.1, 38.5, 49.3 (2C) 78.4, 176.5 (2C). HR-MS m/z:
(calcd for C16H25NO3: 279.18344).
4.1.11. 4-Decyl-10-oxa-4-azatricyclo[5.2.1]decane-3,5-
dione (16). Isolated as a white solid, 68%, mp 30–
1
31 ꢁC. H NMR (CDCl3): d 0.88 (3H, t, J = 6.7 Hz),
1.26 (14H, s), 1.52–1.59 (4H, m, 4H), 1.82–1.85 (2H,
m), 2.81 (2H, s), 3.44 (2H, t, J = 7.3 Hz), 4.84 (2H, t,
J = 2.1 Hz). 13C NMR (CDCl3): d 14.0, 22.7, 26.7,
27.6, 28.7 (2C), 29.1, 29.3, 29.5, 32.0, 39.2, 50.5 (2C),
79.1 (2C), 177.2 (2C). HR-MS m/z: (calcd for
C18H29NO3: 307.21474).
4.1.4. 4-Ethyl-10-oxa-4-azatricyclo[5.2.1]decane-3,5-dione
1
(9). Isolated as a white solid, 81%, mp 166–169 ꢁC. H
NMR (DMSO-d6): 1.09 (3H, t, J = 7.21 Hz), 1.55 (2H,
m), 1.80 (2H, m), 2.80 (2H, s), 3.46 (2H, q, J = 7.1 Hz),
4.81 (2H, dd, J = 2.4, 3.1 Hz). 13C NMR (DMSO-d6):
12.9, 28.5, 33.9, 49.9, 79.0, 176.9. HR-MS m/z: (calcd
for C11H15NO3: 209.10519).
4.1.12. 4-Dodecyl-10-oxa-4-azatricyclo[5.2.1]decane-3,5-
dione (17). Isolated as a white solid, 82%, mp 35–37 ꢁC. 1
H NMR (CDCl3): d 0.87 (4H, m), 1.24 (20H, s), 1.85–
4.1.5. 4-Propyl-10-oxa-4-azatricyclo[5.2.1]decane-3,5-dione
1.82 (2H, m), 1.60–1.50 (4H, m), 2.83 (2H, s), 3.44
1
13
(10). Isolated as a white solid, 80%, mp 79–80 ꢁC. H
(2H, t, J = 7.3 Hz), 4.85 (2H, q, J = 2.3 Hz).
C
NMR (CDCl3): d 0.87 (3H, t, J = 7.3 Hz), 1.55–1.61
(4H, m), 1.83–1.86 (2H, m), 2.84 (2H, s, 2H), 3.44 (2H,
t, J = 7.3 Hz), 4.86 (2H, br s). 13C NMR (CDCl3): d
NMR (CDCl3): d 13.5, 22.1, 26.1, 27.0, 28.0, 28.5,
28.8, 28.9, 29.0, 38.5, 49.3, 78.5, 176.7. HR-MS m/z:
(calcd for C20H33NO3: 335.24604).

6132
T. A. Hill et al. / Bioorg. Med. Chem. 15 (2007) 6126–6134
4.1.13. 4-Tetradecyl-10-oxa-4-azatricyclo[5.2.1]decane-
3,5-dione (18). Isolated as a white solid, 79%, mp 45–
46 ꢁC. H NMR (CDCl3): d 0.86 (2H, m), 1.23 (20H,
4.1.19. 4-(6-Hydroxyhexyl)-10-oxa-4-azatricyclo[5.2.1]-
decane-3,5-dione (26). Isolated as a white solid, 62%,
mp 56–57 ꢁC. H NMR (CDCl3): d 1.27 (4H, m), 1.48
1
1
s), 1.56 (2H, m), 1.83 (2H, quin, J = 4.5 Hz), 2.82 (2H,
s), 3.42 (2H, t, J = 7.3 Hz), 4.84 (2H, q, J = 2.2 Hz).
13C NMR (CDCl3): d 13.5, 22.1, 26.1, 28.0, 28.5, 29.0,
29.1, 38.5, 49.3, 78.5, 176.7. HR-MS m/z: (calcd for
C22H37NO3: 363.27734).
(2H, m), 1.57 (2H, m), 2.15 (2H, m), 2.82 (2H, s), 3.41
(2H, m), 3.54 (2H, m), 4.80 (2H, m). 13C NMR (CDCl3):
d 24.5, 25.6, 26.8, 27.9, 31.8, 38.3, 49.3, 61.9, 78.5, 176.8.
HR-MS m/z: (calcd for C14H21NO4: 267.14706).
4.1.20. 4-(2-Hydroxy-1-methylethyl)-10-oxa-4-azatricy-
clo[5.2.1]decane-3,5-dione (27). Isolated as a yellow solid,
22%, mp 104–107 ꢁC. H NMR (DMSO-d6): 1.28 (3H,
d, J = 7.1 Hz), 1.58 (2H, m), 1.84 (m), 2.14 (1H, s),
2.83 (2H, s), 3.70 (1H, m), 3.80 (1H, m), 4.26 (1H, m),
4.84 (2H, m). 13C NMR (DMSO-d6): 13.8, 28.5, 28.6,
49.6, 49.7, 50.4, 63.5, 79.3, 177.9. HR-MS m/z: (calcd
for C11H15NO4: 225.10011).
4.1.14. 4-Octadecyl-10-oxa-4-azatricyclo[5.2.1]decane-
3,5-dione (19). Isolated as a white solid, 80%, mp 62–
64 ꢁC. 1H NMR (CDCl3): d 1.58 (2H, m), 1.83–1.91
(4H, m), 2.86 (2H, s), 2.33 (2H, t, J = 7.4 Hz), 3.54
(2H, t, J = 6.8 Hz), 4.86 (2H, q, J = 2.3 Hz). 13C NMR
(CDCl3): d 22.0, 28.0, 30.4, 37.5, 49.3, 78.5, 176.7,
177.1. HR-MS m/z: (calcd for C26H45NO3: 419.33994).
1
4.1.15. 4-Allyl-10-oxa-4-azatricyclo[5.2.1]decane-3,5-dione
(20). Isolated as a white solid, 75%, mp 116–117 ꢁC. H
4.1.21. Synthesis of 4-(2-hydroxy-1,1-dimethylethyl)-10-
oxa-4-azatricyclo[5.2.1.02,6]decane-3,5-dione (28). Iso-
lated as a pale yellow solid, 46%, mp 134–136 ꢁC. H
NMR (DMSO-d6): d 1.39 (6H, s), 1.52 (2H, m), 1.78
(2H, m), 2.72 (2H, s), 3.52 (1H, br s), 3.71 (2H, s),
4.78 (2H, dd, J = 2.3, 3.1 Hz). 13C NMR (DMSO-d6):
d 22.1, 28.4, 49.6, 63.0, 68.9, 79.5, 179.1. HR-MS m/z:
(calcd for C12H17NO4: 239.11576).
1
1
NMR (CDCl3): d 1.55–1.62 (2H, m), 1.85–1.88 (2H, m),
2.89 (2H, s), 4.08 (2H, dd, J = 4.0, 1.3 Hz), 4.88–4.90
(2H, m), 5.16–5.22 (2H, m), 5.71–5.76 (1H, m). 13C
NMR (DMSO-d6): d 28.0 (2C), 40.3, 49.4, 78.4, 116.9,
129.8, 176.0 (2C). HR-MS m/z: (calcd for C11H13NO3:
207.08954).
4.1.16. 4-But-3-enyl-10-oxa-4-azatricyclo[5.2.1]decane-3,5-
dione (21). Isolated as a white solid, 31%, mp 64–65 ꢁC.
1H NMR (DMSO-d6): d 1.61 (4H, br s), 2.17 (2H, q,
J = 6.9 Hz), 3.00 (2H, s), 3.37 (2H, t, J = 6.9 Hz), 4.66
(2H, s), 4.99 (2H, m), 5.65 (1H, m). 13C NMR (DMSO-
d6): d 28.8 (2C), 32.2, 38.2, 50.2 (2C), 79.2, 79.2, 117.9,
135.5, 170.1 (2C). HR-MS m/z: (calcd for C12H15NO3:
221.10519).
4.1.22. Synthesis of 4-(1-hydroxymethylpropyl)-10-oxa-4-
azatricyclo[5.2.1]decane-3,5-dione (29). Isolated as a pale
yellow oil, 49%. H NMR (DMSO-d6): d 0.79 (3H, t,
J = 7.4 Hz), 1.38–1.79 (6H, m), 2.81 (2H, q, J = 11.6,
7.0 Hz), 3.01 (1H, br s), 3.63 (1H, m), 3.87 (1H, m),
4.05 (1H, m), 4.79 (2H, s). 13C NMR (DMSO-d6): d
10.3, 20.7, 28.4, 28.5, 49.4, 49.6, 56.5,62.1, 79.2, 178.2.
HR-MS m/z: (calcd for C12H17NO4: 239.11576).
1
4.1.17. 4-Oxiranylmethyl-10-oxa-4-azatricyclo[5.2.1]dec-
ane-3,5-dione (22). m-Chloroperbenzoic acid (2.15 g,
77% in water, 9.66 mmol) was added in one portion to
cooled and magnetically stirred solution at 0 ꢁC of al-
lyl-N-norcantharimide 20 (1.0 g, 4.83 mmol) in CH2Cl2
(20 mL). The resulting solution was warmed to room
temperature, stirred for 16 h before being diluted with
CH2Cl2 (30 mL) and washed with NaHCO3 (3·
10 mL, sat solution). The organic layer was dried
(Na2SO4), filtered and concentrated under reduced pres-
sure to afford a white solid. Flash chromatography (50%
4.1.23. 4-(3,5-Dioxo-10-oxa-4-azatricyclo[5.2.1]dec-4-yl)-
butyric acid (30). Isolated as a white solid, 45%, mp 162–
164 ꢁC. H NMR (CDCl3): d 1.58 (2H, m), 1.83–1.91
(4H, m), 2.33 (2H, t, J = 7.4 Hz), 2.86 (2H, s), 3.54
(2H, t, J = 6.8 Hz), 4.86 (2H, q, J = 2.3 Hz). 13C NMR
(CDCl3): d 22.0, 28.0, 30.4, 37.5, 49.3, 78.5, 176.7,
177.1. HR-MS m/z: (calcd for C12H15NO5: 253.09502).
1
4.1.24. 6-(3,5-Dioxo-10-oxa-4-azatricyclo[5.2.1]dec-4-yl)-
hexanoic acid (31). Isolated as a white solid, 56%, mp
1
110–112 ꢁC. H NMR (CDCl3): d 1.30 (2 H, m), 1.60
EtOAc/Hexane) afforded the norcantharimide epoxide
H
(6H, m), 1.83 (2H, m), 2.32 (2H, t, J = 7.44), 2.86 (2H,
s), 3.45 (2H, t, J = 7.23), 4.86 (2H, q, J = 2.2 Hz). 13C
NMR (CDCl3): d 23.5, 25.4, 26.6, 28.0, 33.2, 38.2,
49.3, 78.5, 176.8, 178.6. HR-MS m/z: (calcd for C1
4H19NO5: 281.12632).
1
(820 mg) as a white solid, 76%, mp 83–84 ꢁC.
NMR (CDCl3): d 1.53–1.59 (2 H, m), 1.79–1.82 (2H,
m), 2.51–2.54 (1H, m), 2.68 (1H, t, J = 4.1 Hz), 2.86
(2H, s), 3.04–3.06 (1H, m), 3.55 (1H, dd, J = 14,
4.6 Hz), 3.64 (1H, dd, J = 14, 4.6 Hz), 4.80–4.82 (2H,
m). 13C NMR (CDCl3): d 27.9 (2C), 39.8 (2C), 45.2,
47.8, 49.3, 49.4 (2C) 78.4, 176.3 (2C). HR-MS m/z:
(calcd for C11H13NO4: 223.08446).
4.1.25. 4-(2,3-Dihydroxypropyl)-10-oxa-4-azatricyclo[5.2.1]-
decane-3,5-dione (23). OsO4 (120 mg of a 2.5% solution in
t-BuOH, 0.012 mmol, 0.5 mol%) was added dropwise to a
magnetically stirred solution of allyl substituted 20
4.1.18. 4-(3-Hydroxypropyl)-10-oxa-4-azatricyclo[5.2.1]-
decane-3,5-dione (25). Isolated as a white solid, 80%, mp
164–166 ꢁC. 1H NMR (CDCl3): d 1.61 (2H, m), 1.84 (2H,
m), 2.89 (2H, s), 3.01 (1H, br s), 3.64 (2H, q, J = 3.6 Hz),
3.70 (2H, t, J = 4.8 Hz), 4.86 (2H, q, J = 2.1 Hz). 13C
NMR (CDCl3): d 27.9, 41.3, 49.4, 59.5, 78.6, 177.1. HR-
MS m/z: (calcd for C10H13NO4: 211.08446).
(500 mg,
2.41 mmol),
N-methylmorpholine-N-oxide
(310 mg, 2.65 mmol) in acetone/water (5:2 mL). The
resulting solution was heated at 80 ꢁC for 16 h before
being diluted with ether (100 mL) and washed with water
(3· 20 mL). The organic layer was concentrated under
reduced pressure to afford a brown solid. Flash chromato-
graphy (70% EtOAc/hexanes) afforded a pale white solid

T. A. Hill et al. / Bioorg. Med. Chem. 15 (2007) 6126–6134
6133
which was recrystallised from EtOAc to afford a white
crystalline solid, 34%. H NMR (DMSO-d6): d 1.62 (4H,
s), 3.00 (2H, s), 3.23–3.32 (2H, m), 3.63 (1H, q,
J = 5.7 Hz), 4.51 (1H, t, J = 5.7 Hz), 4.66 (2H, s), 4.73
(1H, d, J = 4.6 Hz). 13C NMR (DMSO-d6): 27.8 (2C),
41.8, 49.3 (2C) 63.9, 67.9, 78.3 (2C), 177.4. HR-MS m/z:
(calcd for C11H15NO5: 241.09502).
3.01 (2H, s), 4.98 (2H, q, J = 0.9 Hz), 6.86 (2H, d,
J = 8.1 Hz), 7.11 (2H, d, J = 8.1 Hz). 13C NMR
(DMSO-d6): d 28.1 (2C), 49.4, 78.9, 115.4 (2C), 127.4
(2C), 181.5 (2C). HR-MS m/z: (calcd for C14H13NO4:
259.08446).
1
4.1.32. 4-(4-Nitrophenyl)-10-oxa-4-azatricyclo[5.2.1]dec-
ane-3,5-dione (37). Isolated as an orange yellow
1
4.1.26. 4-(3-Hydroxy-2-methoxypropyl)-10-oxa-4-azatri-
cyclo[5.2.1]decane-3,5-dione (24). (1S)-(+)-10-Camphor-
sulfonic acid (15 mg, 0.06 mmol) was added to a
magnetically stirred solution of epoxide 22 (220 mg,
0.98 mmol) in methanol (4 mL). The resulting solution
was warmed to 35 ꢁC and stirred for 16 h before being
concentrated under reduced pressure. The resulting clear
oil was subjected to flash chromatography (70% EtOAc/
Hexanes) to afford a white solid, 78%, mp 95–96 ꢁC. 1H
NMR (DMSO-d6): d 1.60–1.63 (2H, m), 1.85–1.88 (2H,
m), 2.91 (2H, s), 3.33–3.39 (2H, m), 3.38 (3H, s), 3.60–
3.68 (2H, m), 3.94–3.96 (1H, m), 4.88–4.89 (2H, m).
13C NMR (DMSO-d6): d 28.0 (2C), 41.7, 49.4 (2C),
58.7, 67.7, 73.5, 78.6 (2C), 177.0 (2C). HR-MS m/z:
(calcd for C12H17NO5: 255.11067).
solid,76%, mp 207–209 ꢁC. H NMR (CDCl3): d 1.66–
1.69 (2H, m), 1.90–1.94 (2H, m), 3.07 (2H, s), 4.99
(2H, q, J = 0.9 Hz), 7.55 (2H, d, J = 7.1 Hz), 8.29 (2H,
d, J = 7.1 Hz). 13C NMR (CDCl3): d 28.0 (2C), 49.5
(2C), 79.1, 123.7, 126.4, 136.8, 146.5, 174.9. HR-MS
m/z: (calcd for C14H12N2O5: 288.07462).
4.1.33. 4-(3,5-Dioxo-10-oxa-4-azatricyclo[5.2.1]dec-4-yl)
benzoic acid (38). Isolated as a white solid, 36%, mp 275–
1
277 ꢁC. H NMR (CDCl3): 1.63 (2H, m), 1.88 (2H, m),
3.00 (2H, s), 4.96 (2H, q, J = 1.9 Hz), 7.30 (2H, d,
J = 8.1 Hz), 8.13 (2H, d, J = 8.4 Hz). 13C NMR
(CDCl3): 28.0, 49.5, 78.9, 125.3, 129.8, 132.2, 134.3,
160.1, 175.3. HR-MS m/z: (calcd for C15H13NO5:
287.07937).
4.1.27. Synthesis of 4-morpholin-4-yl-10-oxa-4-azatricy-
clo[5.2.1]decane-3,5-dione (32). Isolated as a yellow solid,
43%, mp 169–171 ꢁC. 1H NMR (DMSO-d6): d 1.53 (2H,
m), 1.78 (2H, m), 2.73 (2H, s), 3.18 (4H, t, J = 4.5 Hz),
3.71 (4H, t, J = 4.7 Hz), 4.78 (2H, m); 13C NMR
(DMSO-d6): d 28.5, 47.9, 51.2, 66.7, 79.1, 175.1. HR-
MS m/z: (calcd for C12H16N2O4: 252.1101).
4.1.34. 4-Benzyl-10-oxa-4-azatricyclo[5.2.1]decane-3,5-
dione (39). Isolated as a white solid, 91%, mp 97–
100 ꢁC. 1H NMR (DMSO-d6): d 1.21 (2H, m), 1.54
(2H, m), 1.77 (2H, m), 2.81 (2H, s), 4.56 (2H, s), 4.81
(2H, m), 7.22 (5H, m). 13C NMR (DMSO-d6): d 28.4,
42.3, 49.8, 78.9, 127.5, 127.8, 128.4, 135.3, 176.7. HR-
MS m/z: (calcd for C16H17NO4: 287.11576).
4.1.28. Synthesis of 4-(2-morpholin-4-ylethyl)-10-oxa-4-
azatricyclo[5.2.1.02,6]decane-3,5-dione (33). Isolated as
an orange brown solid, 37%, mp 109–111 ꢁC; 1H
NMR (DMSO-d6): 1.56 (2H, m), 1.80 (2H, m), 2.43-
2.48 (6H, m), 2.84 (2H, s), 3.55 (2H, t, J = 4.5 Hz),
3.60 (4H, t, J = 4.5 Hz), 4.82 (2H, m). 13C NMR
(DMSO-d6): 28.6, 36.6, 49.9, 53.4, 55.1, 67.0, 79.0,
177.1. HR-MS m/z: (calcd for C14H20N2O4: 280.14231).
4.1.35. 4-(4-Methoxybenzyl)-10-oxa-4-azatricyclo[5.2.1]-
decane-3,5-dione (40). Isolated as a white solid, 82%, mp
83–85 ꢁC. H NMR (CDCl3): d 1.60 (2H, m), 1.85 (2H,
m), 2.84 2H, s), 3.77 (3H, s), 4.56 (2H, s), 4.87 (2H, q,
J = 2.1 Hz), 6.81 (2H, d J = 6.7 Hz), 7.25 (2H, d,
J = 6.6 Hz). 13C NMR (CDCl3): d 28.0, 41.4, 49.5,
54.6, 78.5, 113.4, 127.3, 129.1, 158.7, 176.2. HR-MS
m/z: (calcd for C16H17NO4: 287.11576).
1
4.1.29. Synthesis of 4-(3-morpholin-4-ylpropyl)-10-oxa-4-
azatricyclo[5.2.1.02,6]decane-3,5-dione (34). Isolated as
an orange/brown oil, 7%. 1H NMR (DMSO-d6): d
1.55 (2H, m), 1.70 (2H, s), 1.81 (2H, m), 2.81 (2H, s),
2.30 (2H, t), 2.37 (4H, m), 3.48 (2H, t, J = 4.5 Hz),
3.65 (4H, t, J = 4.7 Hz), 4.82 (2H, m). 13C NMR
(DMSO-d6): d 24.2, 28.5, 37.2, 49.9, 51.3, 53.4, 55.8,
66.8, 79.0, 177.2. HR-MS m/z: (calcd for C15H22N2O4:
294.15796).
4.1.36. 4-(3,4-Dimethoxybenzyl)-10-oxa-4-azatricyclo[5.2.1]-
decane-3,5-dione (41). Isolated as a white solid, 76%, mp 155–
157 ꢁC. 1HNMR(CDCl3): d1.60 (2H, m), 1.84 (2H, m), 2.86
(2H, s), 3.83 (3H, s), 3.84 (3H, s), 4.56 (2H, s), 4.87 (2H, q,
J = 2.4 Hz), 6.77 (1H, m), 6.89 (2H, m). 13C NMR (CDCl3):
d 28.0, 41.7, 49.5, 55.3, 78.5, 110.7, 110.9, 120.1, 127.6, 148.1,
148.5, 176.3. HR-MS m/z: (calcd for C17H19NO5:
317.12632).
4.1.37.
4-(2-Aminobenzyl)-10-oxa-4-azatricyclo[5.2.1]-
4.1.30. 4-Phenyl-10-oxa-4-azatricyclo[5.2.1]decane-3,5-
dione (35). Isolated as a bone coloured solid, 79%, mp
171–172 ꢁC. H NMR: (CDCl3): d 1.63–1.65 (2H, m),
1.87–1.90 (2H, m), 3.01 (2H, s), 4.97 (2H, q,
J = 1.1 Hz), 7.25–7.28 (2H, m), 7.42-7.45 (2H, m). 13C
NMR: (CDCl3): 28.1 (2C), 49.5 (2C), 78.9, 125.0 (2C),
128.1, 128.5, 175.7. HR-MS m/z: (calcd for
C14H13NO3: 243.08954).
decane-3,5-dione (42). Isolated as a yellow solid, 43%,
mp 159–160 ꢁC. 1H NMR (DMSO-d6): d 1.54–1.46
(4H, m), 2.74 (2H, s), 3.84 (2H, s), 4.71 (2H, s), 6.54
(1H, t), 6.68 (1H, d), 7.09–7.00 (2H, m). 13C NMR
(DMSO-d6): d 29.0, 53.7, 79.8, 115.3, 116.1, 118.7,
128.9, 129.8, 146.8, 174.1. HR-MS m/z: (calcd for
C15H16N2O3: 272.11609).
1
4.1.38. Bis-3,6-epoxycyclohexane-1,2-dicarboximido)-tri-
methylene (bis-norcantharimide-propyl linker) (43). Iso-
lated as a white solid, 80%. H NMR (DMSO-d6): d
1.58 (2H, quin), 1.85 (4H, m), 2.86 (2H, s), 3.43 (2H, m),
4.1.31. 4-(4-Hydroxyphenyl)-10-oxa-4-azatricyclo[5.2.1]-
decane-3,5-dione (36). Isolated as a white solid, 45%,
172–173 ꢁC. H NMR (CDCl3): d 1.56–1.62 (4H, m),
1
1

6134
T. A. Hill et al. / Bioorg. Med. Chem. 15 (2007) 6126–6134
4.86 (2H, m). 13C NMR (DMSO-d6): d 24.7, 28.0, 35.6,
49.4, 78.5, 176.5. HR-MS m/z: (calcd for C19H22N2O6:
374.14779).
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1
Isolated as a white solid, 62%. H NMR (CDCl3): d
1.22 (10H, s), 1.59–1.51 (4H, m), 1.85 (2H, m), 2.84
(2H, s), 3.43 (2H, t, J = 7.3 Hz), 4.85 (2H, q,
J = 2.2 Hz). 13C NMR (CDCl3): d 26.0, 27.0, 28.5, 28.8,
28.9, 38.5, 49.3, 78.5, 176.6. HR-MS m/z: (calcd for
C28H40N2O6: 500.28864).
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Acknowledgment
This work received financial support from the Hunter
Medical Research Institute and the Biotechnology Inno-
vation Fund, AusIndustry, Australia.
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References and notes
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