Norcantharimide analogues possessing terminal phosphate esters and their anti-cancer activity

Article abstract of DOI:10.1016/j.bmc.2011.01.031

A family of norcantharidin analogues possessing a terminal alcohol (ethanol, propanol, butanol, pentanol, hexanol and cyclohexanol) moiety were treated with either chlorodiethyl, chlorodiphenyl or chloro-bis-trichloroethyl- phosphate to afford highly focused libraries of the corresponding phosphate esters. Subsequent biological screening against a panel of nine human cancer cell lines identified a trend between the ease of phosphate unmasking (phosphate ester hydrolysis) and cell death. The most potent analogues possessed either a diphenyl or a bis-trichloroethyl moiety. The effect of alkyl spacer was also examined with the hexyl analogues typically more potent. 4-Aza-4-(3-{bis(2,2,2- trichloroethyl)phosphate}propyl)-10-oxatricyclo[5.2.1.0]decane-3,5-dione (10b) was the most potent analogue synthesised with an average GI₅₀ of 11 μM across a panel of nine human carcinoma cell lines: colon carcinoma (HT29 and SW480); breast carcinoma (MCF-7); ovarian carcinoma (A2780); lung carcinoma (H460); skin carcinoma (A431); prostate carcinoma (DU145); neuronal carcinoma (BE2-C) and brain carcinoma (SJ-G2). This represents a fivefold improvement in anti-proliferative activity relative to the lead, norcantharidin.

Full text of DOI:10.1016/j.bmc.2011.01.031

Bioorganic & Medicinal Chemistry 19 (2011) 5734–5741
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Norcantharimide analogues possessing terminal phosphate esters
and their anti-cancer activity
Mark J. Robertson a, Christopher P. Gordon a, Jayne Gilbert b, Adam McCluskey a, , Jennette A. Sakoff b
⇑
a Department of Chemistry, School of Environmental and Life Science, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia
b Department of Medical Oncology, Calvary Mater Newcastle Hospital, Edith Street, Waratah, NSW 2298, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
A family of norcantharidin analogues possessing a terminal alcohol (ethanol, propanol, butanol, penta-
nol, hexanol and cyclohexanol) moiety were treated with either chlorodiethyl, chlorodiphenyl or
chloro-bis-trichloroethyl-phosphate to afford highly focused libraries of the corresponding phosphate
esters. Subsequent biological screening against a panel of nine human cancer cell lines identified a
trend between the ease of phosphate unmasking (phosphate ester hydrolysis) and cell death. The most
potent analogues possessed either a diphenyl or a bis-trichloroethyl moiety. The effect of alkyl spacer
was also examined with the hexyl analogues typically more potent. 4-Aza-4-(3-{bis(2,2,2-trichloroeth-
yl)phosphate}propyl)-10-oxatricyclo[5.2.1.0]decane-3,5-dione (10b) was the most potent analogue syn-
Received 30 June 2010
Revised 12 January 2011
Accepted 16 January 2011
Available online 1 February 2011
Keywords:
Cantharidin
Norcantharidin
Norcantharimide
Cytotoxicity
Anti-cancer
thesised with an average GI50 of 11 lM across a panel of nine human carcinoma cell lines: colon
carcinoma (HT29 and SW480); breast carcinoma (MCF-7); ovarian carcinoma (A2780); lung carcinoma
(H460); skin carcinoma (A431); prostate carcinoma (DU145); neuronal carcinoma (BE2-C) and brain
carcinoma (SJ-G2). This represents a fivefold improvement in anti-proliferative activity relative to the
lead, norcantharidin.
Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
1. Introduction
Numerous research groups have reported various structural
modifications of norcantharidin, ones in which protein phospha-
Cantharidin (1) and norcantharidin (2) have a long history of use
as anti-proliferative agents (Fig. 1).1 This use originates from the
Chinese traditional medicine, Mylabris from the dried bodies of blis-
ter beetles, of which cantharidin is the active component. Mylabris
has been utilized in the treatment of various cancerous growths and
is reported as a safe and effective treatment of molluscum contagio-
sum.2–4 Cantharidin is a small molecule amenable to rapid struc-
tural modifications; lipophilic not requiring an active transport
mechanism to enter cells; it is not a substrate for p-glycoprotein;
it does not induce myelosuppression and indeed has been shown
to induce haematopoiesis in animal and human studies. However
cantharidin does possess liver and renal toxicities.5–12 Fortunately,
the removal of the 2,3-dimethyl substituents furnishing norcantha-
ridin in addition to simplifying the chemistry, appears to overcome
these toxicity issues. Norcantharidin is reported to induce apoptosis
in human cervical,13,14 oral,15,16 melanoma,17 ginival,15 bladder,18
adenocystic carcinoma,19 glioblastoma,20 bone,21 leukaemia,21
ovarian,22 liver,23,24,21 gall bladder25 and colon26–29 cancer cell lines,
however so far the main application is for hepatoma.30,31
tase inhibition is maintained and others in which this activity is re-
moved, but anti-proliferative activity is maintained.2–4,29,31–43 As is
routine in our laboratory we conducted both protein phosphatase
1 and 2A inhibition studies, and in keeping with our earlier reports
relating to similar cantharimides, all analogues herein were devoid
of any protein phosphatase inhibitory properties. These canthari-
mide analogues are exemplified by the generic (3). In our own
laboratory we have developed a series of bis-norcantharidimides
with modest levels of activity, and N-aromatic variants with poor
to modest activity. N-aliphatic and N-aliphatic-functional
analogues are much less studied. Indeed in our most recent report
we noted that that introduction of a free terminal –OH moiety (4)
gave rise to a modest level of cytotoxicity (Fig. 2).43
O
O
O
O
O
O
O
O
1
2
⇑
Corresponding author. Tel.: +61 249 216486; fax: +61 249 215472.
E-mail address: Adam.McCluskey@newcastle.edu.au (A. McCluskey).
Figure 1. Chemical structures of cantharidin and norcantharidin.
0968-0896/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.01.031

M. J. Robertson et al. / Bioorg. Med. Chem. 19 (2011) 5734–5741
5735
Interestingly in subsequent SAR development, modifications of
O
O
the terminal –OH were unsuccessful in increasing anti-prolifera-
tive activity. Methylation of the C20-OH abolished what little
activity that was present, whilst isosteric modifications of –OH to
–COOH, morpholino, ethylmorpholino, etc. all proved ineffective.43
Notwithstanding this, the fact that the incorporation of a second
norcantharidin unit did increase activity suggested that modifica-
tions of this N-R substituent was possible. Herein we report an ele-
gantly simple series of modifications that furnish considerable
enhancements in anti-proliferative activity.
OH
OH
O
O
N
N
O
O
7b
7a
Figure 3. Chemical structures of N-alcohol substituted lead norcantharimides 7a
and 7b.
modest reduction of the anti-proliferative activity noted with 8a
(average GI50 values of 47 and 29 lM respectively).
2. Results
Having established the favourable outcome with the diethyl
phosphate analogues, we next turned our attention to variations
of the phosphate ester moiety. We thus examined the commer-
cially available diphenyl (9) and trichloroethyl (10) phosphates.
On examination of the cytotoxicity screening outcomes (Table 2)
it is clear that the diphenylphosphates (9a–f) on the whole display
an improved anti-proliferative profile. Only the short alkyl spacer
analogues (9a and 9c) are bereft of activity. The butyl (9d), pentyl
(9e) and hexyl (9b) display activities varying from equipotent with
2 to a fivefold improvement returning average GI50 values of 24, 48,
Our current investigations commenced with the synthesis of
the N-ethanol and N-hexanol analogues (7a, X = CH2CH2;
R1 = OH; 7b X = CH2(CH2)5; R1 = OH; Fig. 3) as shown in and
Scheme 1.
As anticipated screening against our panel of cancer cell lines
afforded no activity (GI50 >100 lM) in all cases. As we have previ-
ously shown that simple modifications of this terminal group did
not increase activity, but the introduction of a second group (OH)
did, we decided to explore the introduction of a more highly func-
tionalized end group. At this time we were also exploring the intro-
duction of phosphates and related groups in an allied program, and
hence it seemed logical to explore a similar series of analogues
here. Thus, treatment of 7a,b with diethylchlorophosphate in the
presence of n-butyltin oxide and triethylamine gave the desired
terminally substituted phosphate analogue (8a, X = CH2CH2;
R1 = P(O)(OEt)2 and 8b, X = –(CH2)6–; R1 = P(O)(OEt)2).
and 12.5
almost twice as potent as the parent 2 with an average GI50 of
27 M. Only the ethyl spacer based trichloroethyl phosphate
lM respectively. The 1,2-disubstituted cyclohexane (9f) is
l
(10a) analogue, in this family, is inactive with all others (10b–f)
returning modest to good anti-proliferative activity. The most po-
tent analogue in this last series is the hexyl spacer (10b, average
GI50 = 11
the butyl (10d, average GI50 = 24
hexyl (10f, average GI50 = 55 M) and the propyl (10c, average
GI50 = 67 M). Excepting 10a and 10c, all trichloroethyl phosphate
lM), followed by the pentyl (10e, average GI50 = 20
lM),
As can be seen from Table 1, the introduction of the terminal
diethyl phosphate to 7a with the synthesis of 8a brought immedi-
lM), the 1,2-disusbstituted cyclo-
l
ate rewards with an increase from no activity at 100
lM drug con-
l
centration to an average GI50 value of 29 M across the nine
l
analogues are more potent than 2. Interestingly, the hexyl spacer
analogue of the diphenyl and trichloroethyl series displayed com-
parable anti-proliferative activity between the two series and the
greatest potency within each of these series.
carcinoma cell lines examined. This represents an improvement
over the parent norcantharidin across the same panel of cancer cell
lines (average GI50 = 42 lM) and equipotent with the most potent
of our previously reported nocantharimides (bis-3,6-epoxycyclo-
The results shown in Table 2 display two clear trends. The first
is that with the exception of the small linker (ethyl 8a and cyclo-
hexyl 8f) analogues the ethyl ester protection results in a lack of
bioactivity. This in itself is not surprising. Simple alkyl esters are
more resistant to hydrolysis than aromatic or trihaloalkyl phos-
phates and so it may be expected that the possibly bioactive
‘naked’ phosphate form of the molecule is not reached.44 In general
both the phenyl and bis(trichloroethyl) analogues show far better
bioactivity relative to the simple alkyl ester analogues which can
be explained by this ease of hydrolysis.45,46 Why the shorter chain
leads to a increase in anti-proliferative activity is not clear, espe-
cially as is the case of the ethyl linker, where the diphenyl (9a)
and bis(trichloroethyl) (10a) show no discernable activity. The sec-
ond trend is the effect of linker length. For both the diphenyl (9)
and bis(trichloroethyl) (10) series the optimal chain length appears
to lie with the hexyl chain, even though this result is not mirrored
in the diethyl series. In general none of the compounds tested dis-
played any major specificity towards one cancer line over another
suggesting the target is fairly generic to all cancer forms.
hexane-1,2-dicarboximido)-trimethylene (bis-norcantharimide-
propyl linker) average GI50 = 26 l
M).43 Interestingly the same
substitution with the hexyl analogue 8b showed no discernable
increase in anti-proliferative activity, possibly an artefact of the
increased chain length. Hence in this study we sought to investi-
gate the effect of the chain length X and also the nature of the
phosphate ester substituents.
The development of the required focused compound libraries
was as per Scheme 1. Analogues with variations in chain length
X and differentially substituted phosphates were synthesised in
good yield (37–83%) and subsequently evaluated for their anti-pro-
liferative effects. The outcome of this screening is shown in Table 2.
As noted previously, the introduction of a diethylphosphate
moiety confers favourable anti-proliferative properties to 8a, but
continued exploration of the alkyl spacer with this series of com-
pounds through propyl to hexyl (8b–e) renders these analogues
inactive. Introduction of a conformationally restricted short linker
in the guise of a 1,2-disubstituted cyclohexane (8f) results in a
O
O
GI50(MCF-7) = 46 4 µM
O
O
N
R
N
OH
OH
O
O
3
4
Figure 2. Chemical structure of generic cantharimides (3) and a glycerol substituted norcantharimide (4) displaying modest levels of cytotoxicity against the MCF-7 breast
cancer cell line (from Ref. 43).

5736
M. J. Robertson et al. / Bioorg. Med. Chem. 19 (2011) 5734–5741
O
O
O
a: X = -CH2CH2-
b: X = -(CH2)6-
c: X = -(CH2)3-
d: X = -(CH2)4-
e: X = -(CH2)5-
f: X =
(i), (ii)
(iii)
O
+
O
O
O
O
N
X OH
O
O
O
5
6
2
7a - 7f
(iv)
O
N
O
8 R = P(O)(OEt)2
O
X
O
R
9 R = P(O)(OPh)2
10 R = P(O)(OCH2CCl3)2
8a - 10f
Scheme 1. Reagents and conditions: (i) Et2O, rt, 48 h; (ii) H2, 10% Pd–C, rt, EtOH; (iii) H2N–X–OH,
D
, 18 h; (iv) ClP(O)(OR)2, n-Bu2O, Et3N, rt.
Table 1
Anti-proliferative effects (GI50 values in
l
M) of norcantharidin (2), lead N-alcohols (7a, 7b) and the lead phosphate esters (8a, 8b) against a panel of human cancer cell lines
O
O
N
X O
R
O
Compound
X
R
HT29a
57
>100
>100
33 1.0
>100
SW480a
44
>100
>100
18 0.4
>100
MCF-7b
68
>100
>100
28 1.3
>100
A2780c
38
>100
>100
15 0.6
>100
H460d
45
>100
>100
49 5.0
>100
A431e
31
>100
>100
27 1.5
>100
DU145f
28
>100
>100
21 3.3
>100
BE2-Cg
43
SJ-G2h
23
>100
>100
43 4.1
>100
2
—
—
H
H
5
6
4
1
3
1
3
6
3
7a
7b
8a
8b
CH2CH2
–(CH2)6–
CH2CH2
–(CH2)6–
>100
>100
i
P(O)(OEt)2
P(O)(OEt)2
—
>100
A
B
C
D
E
F
Colon carcinoma.
Breast carcinoma.
Ovarian carcinoma.
Lung carcinoma.
Skin carcinoma.
Prostate carcinoma.
Neuronal carcinoma.
Brain carcinoma.
Not determined.
G
H
i
To confirm the importance of the norcantharidin scaffold, an
3. Conclusion
analogue of one of our more active compounds, (9d), having a sim-
ple pthalimido group (12) was synthesised as shown in Scheme 2.
This was subjected to the same biological testing and the results
are shown in Table 3. This phenyl ester was inactive across all cell
lines and clearly shows the crucial influence of the norcantharidin
on anti-proliferative activity.
Phosphate esters have been introduced in the past to increase
bioactivity by either acting as a prodrug.43 The problem therein
is the addition of a ‘naked’ phosphate group, which is negatively
charged, results in a molecule with low membrane permeability.
Masking this group as an ester prodrug can increase cell perme-
ability, affording greater cellular uptake followed by esterase ac-
tion to release the bioactive compound. This effect is the most
likely cause of the increased bioactivity as the other probable fate
of these types of molecules is phosphate cleavage by a phosphatase
enzyme to yield the hydroxyl substituted base scaffolds (7). These
types of molecules have been tested in the past and found to be
inactive.43
A series of norcantharidin (2) analogues containing a pro-
tected phosphate tail at varying distance from the tricylic scaf-
fold were synthesized in four steps in modest yields. Biological
screening of these analogues against a panel of nine human can-
cer cell lines: human colon carcinoma cell lines (HT29 and
SW480); human breast carcinoma cell line (MCF-7); human
ovarian carcinoma cell line (A2780); human lung carcinoma cell
line (H460); human skin carcinoma cell line (A431); human
prostate carcinoma cell line (DU145); human neuroblastoma cell
line (BE2-C); human glioblastoma cell line (SJ-G2) resulted in the
identification of a new series of functionalised norcantharidin
analogues with enhanced broad spectrum anti-proliferative
activity. Multiple analogues are at least equipotent with norcant-
haridin and a number displayed about a fivefold increase in cell
death. The relative ease of phosphate ester hydrolysis correlates
well with the observed anti-proliferative activity. Of the phos-
phates examined the diphenyl and bis-trichloroethyl analogues

M. J. Robertson et al. / Bioorg. Med. Chem. 19 (2011) 5734–5741
5737
Table 2
Anti-proliferative effects (GI50 values in
l
M) of terminal phosphate esters 8a–10f against a panel of human cancer cell lines
O
O
N
X O
R
O
Compound
X
R
HT29a
SW480a
MCF-7b
A2780c
H460d
A431e
DU145f
BE2-Cg
SJ-G2h
i
8a
8b
8c
8d
8e
–CH2CH2–
–(CH2)6–
–(CH2)3–
–(CH2)4–
–(CH2)5–
P(O)(OEt)2
P(O)(OEt)2
P(O)(OEt)2
P(O)(OEt)2
P(O)(OEt)2
33 1.0
>100
—
>100
>100
18 0.4
>100
—
>100
>100
28 1.3
>100
—
>100
>100
15 0.6
>100
—
>100
>100
49 5.0
>100
—
>100
>100
27 1.5
>100
—
>100
>100
21 3.3
>100
—
>100
>100
—
43 4.1
>100
—
>100
>100
>100
i
i
i
i
i
i
i
i
i
—
>100
>100
8f
P(O)(OEt)2
57 1.7
>100
33 3.2
>100
14 0.33
>100
28 1.2
50 1.2
50 3.2
>100
22 2.9
89 1.0
47 2.9
>100
36
3
31 4.5
60 1.7
>100
O R
9a
9b
9c
9d
9e
–CH2CH2–
–(CH2)6–
–(CH2)3–
–(CH2)4–
–(CH2)5–
P(O)(OPh)2
P(O)(OPh)2
P(O)(OPh)2
P(O)(OPh)2
P(O)(OPh)2
>100
11 0.8
>100
17 1.0
46 2.3
>100
25 3.7
>100
64 4.8
63 1.3
>100
7.0 0.2
>100
13 0.6
52 1.0
>100
14 0.3
>100
25 3.2
56 2.1
5.4
0
9.1
1
13
0
14
0
>100
14 0.2
42 3.1
>100
>100
21 1.9
45 2.0
>100
17 2.0
50 2.9
19
1
31 1.3
9f
P(O)(OPh)2
11 1.1
26 1.5
14 0.6
24 1.3
48 3.1
26 0.7
35 1.3
28 0.9
31 1.0
O R
10a
10b
10c
10d
10e
–CH2CH2–
–(CH2)6–
–(CH2)3–
–(CH2)4–
–(CH2)5–
P(O)(OCH2Cl3)2
P(O)(OCH2Cl3)2
P(O)(OCH2Cl3)2
P(O)(OCH2Cl3)2
P(O)(OCH2Cl3)2
>100
11 0.3
>100
10 0.7
>100
12 0.9
>100
7.7 0.3
>100
13 0.3
>100
12 0.3
>100
13 0.9
>100
>100
9.3 0.1
>100
14 0.3
57
4
72
5
60
28
6
0
47
9
78
8
80
7
50
40
3
2
58
3
16 1.5
15 0.9
32 1.7
17 0.3
26 1.0
16 0.9
56 1.0
29 0.7
31 1.9
18 1.0
49 0.3
48 0.3
19 1.7
16 0.2
26
1
22 0.7
10f
P(O)(OCH2Cl3)2
56 9.2
69 8.6
54 12.0
39 6.4
>100
48 4.6
69 8.1
51 7.6
45 5.8
O R
A
B
C
D
E
F
Colon carcinoma.
Breast carcinoma.
Ovarian carcinoma.
Lung carcinoma.
Skin carcinoma.
Prostate carcinoma.
Neuronal carcinoma.
Brain carcinoma.
Not determined.
G
H
i
O
O O
P
O
O
(i)
(ii)
N
O
O
O
N
OH
O
O
O
11
12
Scheme 2. Reagents and conditions: (i) H2N–(CH2)4–OH, toluene,
D, MW, 20 min; (ii) ClP(O)(OPh)2, n-Bu2O, Et3N, rt.
displayed the highest level of cell death. In general the more la-
bile phosphate esters six carbons from the norcantharidin scaf-
fold were the most active. The lack of activity with the
pthalimido analogue (12) demonstrates the pivotal nature of
the norcantharidin skeleton to the observed anti-proliferative
activity.
GCMS was performed using a Shimadzu GCMS-QP2010. The instru-
ment uses a quadrupole mass spectrometer and detects samples
via electron impact ionisation (EI) or chemical ionisation using
methane (CI). Electrospray Mass spectra were recorded on a
Shimadzu LCMS 2010 EV using a mobile phase of 1:1 acetoni-
trile/H2O with 0.1% formic acid.
4.1.1. Cell culture and stock solutions
4. Experimental
Stock solutions were prepared as follows and stored at ꢀ20 °C:
cantharidin (Biomol, USA) as a 30 mM solution in dimethylsulfox-
ide (DMSO); norcantharidin as a 30 mM solution in water and nor-
cantharidin 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, Austra-
lia) supplemented with 10% foetal bovine serum, 10 mM sodium
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 (Bruker Advance 300MX).
bicarbonate, penicillin (100 IU/mL), streptomycin (100
and glutamine (4 mM).
lg/mL),

5738
M. J. Robertson et al. / Bioorg. Med. Chem. 19 (2011) 5734–5741
Table 3
Anti-proliferative comparison (GI50 values in
lM) of the norcantharidin ester 9d versus an analogous phthlamido ester against a panel of human cancer cell lines
Compound
HT29a
SW480a
28 1.2
MCF-7b
A2780c
17 1.0
H460d
A431e
DU145f
BE2-Cg
SJ-G2h
O
O O
P
O
N
O
O
9d
12
14 0.2
19
1
64 4.8
21 1.9
13 0.6
25 3.2
17 2.0
9d
O
O
O O
P
N
O
O
>100
>100
>100
>100
>100
>100
>100
>100
>100
12
O
A
B
C
D
E
Colon carcinoma.
Breast carcinoma.
Ovarian carcinoma.
Lung carcinoma.
Skin carcinoma.
Prostate carcinoma.
Neuronal carcinoma.
Brain carcinoma.
F
G
H
4.1.2. In vitro growth inhibition assay
Cells in logarithmic growth were transferred to 96-well plates.
Cytotoxicity was determined by plating cells in duplicate in
(211 mg, 1 mmol) in anhydrous CH2Cl2 (10 mL) was added n-Bu2S-
nO (50 mg, 0.2 mmol, 0.2 equiv) and the resulting mixture stirred
at room temperature for 30 min under a N2 atmosphere. Next
diethyl chlorophosphate (172.5 mg, 1 mmol, 1.0 equiv) and trieth-
100
(24 h after plating) when the cells were in logarithmic growth,
100 L medium with or without the test agent was added to each
lL medium at a density of 2500–4000 cells/well. On day 0,
ylamine (500 lL, 3.6 mmol, 3.6 equiv) was then added and the
l
mixture stirred for 18 h. The reaction was then quenched with
H2O (10 mL). The organic layer was then separated, dried (MgSO4)
and evaporated to dryness. The crude product was then purified
via flash column chromatography using 4:1 EtOAc/hexanes as the
solvent to yield the title compound (8a) as a white solid (70%),
mp 172–174 °C.
well. After 72 h drug exposure growth inhibitory effects were
evaluated using the MTT (3-[4,5-dimethyltiazol-2-yl]-2,5-diphe-
nyl-tetrazolium bromide) assay and absorbance read at 540 nm.
Percentage growth inhibition was determined at a fixed drug con-
centration of 100 lM. A value of 100% is indicative of total cell
growth inhibition. Those analogues showing appreciable percent-
age 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 values on day 0 and those
at the end of drug exposure.43
1H NMR (CDCl3): d 4.85, dd, J = 3.11, 2.23 Hz, 2H; 4.12, m, 2H;
4.07, m, 4H; 3.75, t, J = 5.36 Hz, 2H; 2.92, s, 2H; 1.85, m, 2H; 1.60,
m, 2H; 1.31, t, J = 7.1 Hz, 6H.
13C NMR (CDCl3): d 176.4, 78.3, 63.5, 62.5, 49.6, 38.5, 28.0,
15.5 ppm.
MS(EI) m/z: 347.
HR-MS (ESI, M+H) m/z: calcd for C14H23NO7P: 348.12121; found
348.12122.
4.2. General synthesis
4.2.2.1.2.
4-Aza-4-(6-hydroxyhexyl)-10-oxatricyclo[5.2.1.0]dec-
4.2.1. General Method A
4.2.1.1. Formation of the N-substituted norcantharidin ana-
ane-3,5-dione (7b). Synthesised as described in Method A from
norcantharidin (2) and 6-aminohexanol to yield the title com-
pound (7b) as a colourless oil (45%).
logues
(7a–f). 4.2.1.1.1. Illustrative synthesis of 4-aza-4-(2-
To
hydroxyethyl)-10-oxatricyclo[5.2.1.0]decane-3,5-dione (7a)43
.
1H NMR (CDCl3): d 4.68, dist t, 2H; 3.40, t, J = 6.5 Hz, 2H; 3.26, t,
J = 7.2 Hz, 2H; 3.08, br s, OH; 2.73, s, 2H; 1.69, m, 2H; 1.45, m, 2H;
1.38, m, 4H; 1.15, m, 4H.
norcantharidin (2) (168 mg, 1 mmol) in toluene (10 mL) was added
2-aminoethanol (67 mg, 1.1 mmol, 1.1 equiv) and triethylamine
(202 mg, 2.0 equiv). The resultant solution was heated at reflux
for 18 h and evaporated to dryness. The desired compound (7a)
was obtained via flash column chromatography using EtOAc as
the solvent as a white solid (73%), mp 164–166 °C.
1H NMR (CDCl3): d 4.86, dd, J = 2.1 Hz, 2H; 3.70, t, J = 4.8 Hz, 2H;
3.64, q, J = 3.6 Hz, 2H; 3.01 br s, OH; 2.89, s, 2H; 1.84, m, 2H; 1.61,
m, 2H.
13C NMR (CDCl3): d 177.2, 78.7, 61.9, 49.5, 38.5, 32.0, 28.2, 27.0,
25.9, 24.8 ppm.
MS(ES) m/z: 268 (M+H).
HR-MS (ESI, M+H) m/z: calcd for C14H22NO4: 268.15488; found
268.15496.
4.2.2.1.3. 4-Aza-4-(3-hydroxypropyl)-10-oxatricyclo[5.2.1.0]dec-
ane-3,5-dione (7c). Synthesised as described in Method A from
norcantharidin (2) and 3-aminopropanol to yield the title com-
pound (7c) as a colourless oil (80%).
13C NMR (CDCl3): 177.1, 78.6, 59.5, 49.4, 41.3, 27.9.
MS(ES) m/z: 212 (M+H).
1H NMR (CDCl3): d 4.83, dist t, 2H; 3.62, t, J = 6.5 Hz, 2H; 3.48, q,
J = 6.5 Hz, 2H; 2.73, s, 2H; 1.82, m, 2H; 1.73, m, 2H; 1.65, m, 2H.
13C NMR (CDCl3): d 177.8, 79.0, 58.5, 49.8, 35.2, 30.2, 28.4 ppm.
MS(ES) m/z: 226 (M+H).
4.2.2. General Method B
4.2.2.1. Formation of the phosphate esters (8a–10f). 4.2.2.1.1.
Illustrative synthesis of 4-aza-4-(2-{diethylphosphate}ethyl)-10-
oxatricyclo[5.2.1.0]decane-3,5-dione (8a). To a solution of 4-aza-
4-(2-hydroxyethyl)-10-oxatricyclo[5.2.1.0]decane-3,5-dione (7a)
HR-MS (ESI, M+H) m/z: calcd for C11H16NO4: 226.10793; found
226.10797.

M. J. Robertson et al. / Bioorg. Med. Chem. 19 (2011) 5734–5741
5739
4.2.2.1.4.
4-Aza-4-(4-hydroxybutyl)-10-oxatricyclo[5.2.1.0]dec-
Method
B
from
4-aza-4-(5-hydroxypentyl)-10-oxatricy-
ane-3,5-dione (7d). Synthesised as described in Method A from
norcantharidin (2) and 4-aminobutanol to yield the title compound
(7d) as a pale yellow oil (61%).
clo[5.2.1.0]decane-3,5-dione (7e) and diethyl chlorophosphate to
yield the title compound (8e) as a clear oil (60%).
1H NMR (CDCl3): d 4.81, br s, 2H; 4.05, m, 4H; 3.95, m, 2H; 3.41,
t, J = 7.2 Hz, 2H; 2.82, s, 2H; 1.78, m, 2H; 1.56, m, 8H; 1.30, t,
J = 7.2 Hz, 6H.
1H NMR (CDCl3): d 4.77, dist t, 2H; 3.54, t, J = 6.3 Hz, 2H; 3.42, t,
J = 6.9 Hz, 2H; 2.80, s, 2H; 2.43, br s, OH; 1.76, m, 2H; 1.55, m, 4H;
1.45, m, 2H.
13C NMR (CDCl3): d 177.1, 79.0, 66.1, 63.6, 49.8, 38.7, 29.6, 28.5,
27.0, 22.5, 16.1 ppm.
13C NMR (CDCl3): d 177.2, 78.9, 61.8, 49.7, 38.6, 29.4, 28.4,
23.9 ppm.
MS(ES) m/z: 390 (M+H).
MS(ES) m/z: 240 (M+H).
HR-MS (ESI, M+H) m/z: calcd for C12H18NO4: 240.12358; found
240.12359.
HR-MS (ESI, M+H) m/z: calcd for C17H29NO7P: 390.16816; found
390.16823.
4.2.2.1.10. 4-Aza-4-(2-{diethylphosphate}cyclohexyl)-10-oxatricy-
clo[5.2.1.0]decane-3,5-dione (8f). Synthesised as described in
4.2.2.1.5. 4-Aza-4-(5-hydroxypentyl)-10-oxatricyclo[5.2.1.0]dec-
ane-3,5-dione (7e). Synthesised as described in Method A from
norcantharidin (2) and 5-aminopentanol to yield the title com-
pound (7e) as a white solid (70%). mp 96–98 °C.
1H NMR (CDCl3): d 4.86, br s, OH; 4.66, s, 2H; 3.54, t, J = 6.5 Hz,
2H; 3.46, t, J = 7.0 Hz, 2H; 3.14, s, 2H; 1.79, m, 2H; 1.72, m, 2H;
1.53, m, 4H; 1.25, m, 2H.
Method
B
from 4-aza-4-(2-hydroxycyclohexyll)-10-oxatricy-
clo[5.2.1.0]decane-3,5-dione (7f) and diethyl chlorophosphate to
yield the title compound (8f) as an off white semi solid (45%).
1H NMR (CDCl3): d 4.72, dist t, 2H; 4.15, m, 4H; 3.83, m, 1H;
3.56, m, 1H; 2.70, s, 2H; 2.18, m, 2H; 1.94, m, 2H; 1.72, m, 2H;
1.49, m, 4H; 1.28, t, J = 7.1 Hz, 6H; 12.3, m, 2H.
13C NMR (75 MHz, CDCl3): d 177.2, 79.0, 69.0, 65.1, 50.6, 49.3,
34.3, 28.4, 26.2, 15.8 ppm.
13C NMR (CDCl3): d 180.3, 79.1, 61.1, 49.3, 38.4, 30.3, 27.4, 26.0,
21.8 ppm.
MS(ES) m/z: 254.1 (M+H).
MS(ES) m/z: 402 (M+H).
HR-MS (ESI, M+H) m/z: calcd for C13H20NO4: 254.13923; found
254.13919.
HR-MS (ESI, M+H) m/z: calcd for C18H29NO7P: 402.16816; found
402.16819.
4.2.2.1.6.
4-Aza-4-(2-hydroxycyclohexyll)-10-oxatricy-
4.2.2.1.11.
4-Aza-4-(2-{diphenylphosphate}ethyl)-10-oxatricy-
clo[5.2.1.0]decane-3,5-dione (7f). Synthesised as described in
Method A from norcantharidin (2) and 2-aminocyclohexanol to
yield the title compound (7f) as a white solid (70%), mp 172–
174 °C.
clo[5.2.1.0]decane-3,5-dione (9a). Synthesised as described in
Method B from 4-aza-4-(2-hydroxyethyl)-10-oxatricyclo[5.2.1.0]
decane-3,5-dione (7a) and diphenyl chlorophosphate to yield the
title compound (9a) as a colourless oil (54%).
1H NMR (CDCl3: d 7.28, m, 4H; 7.11, m, 6H; 4.78, dd, J = 2.7 Hz,
2.3 Hz, 2H; 4.33, dt, J = 8.7, 5.4, 5.5 Hz, 2H; 3.75, t, J = 5.4 Hz, 2H;
2.76, s, 2H; 1.73, m, 2H; 1.47 (m, 2H).
1H NMR (CDCl3): d 4.81, dist t, 2H; 3.91, m, 1H; 3.65, m, 1H;
3.06, br s, OH; 2.76, s, 2H; 2.19, m, 2H; 1.99, m, 2H; 1.79, m, 2H;
1.55, m, 6H.
13C NMR (CDCl3): d 177.3, 79.2, 69.5, 50.8, 49.6, 45.9, 34.6, 28.6,
26.4, 8.6 ppm.
13C NMR: d 176.4, 149.8, 129.3, 124.9, 119.5, 78.3, 63.9, 49.4,
38.2, 29.7 ppm.
MS(ES) m/z: 266 (M+H).
MS(ES) m/z: 444 (M+H).
HR-MS (ESI, M+H) m/z: calcd for C14H20NO4: 266.13923; found
266.13940.
HR-MS (ESI, M+H) m/z: calcd for C22H23NO7P: 444.12121; found
444.12119.
4.2.2.1.7.
clo[5.2.1.0]decane-3,5-dione (8b). Synthesised as described in
Method from 4-aza-4-(6-hydroxyhexyl)-10-oxatricy-
4-Aza-4-(6-{diethylphosphate}hexyl)-10-oxatricy-
4.2.2.1.12.
4-Aza-4-(6-{diphenylphosphate}hexyl)-10-oxatricy-
clo[5.2.1.0]decane-3,5-dione (9b). Synthesised as described in
Method B from 4-aza-4-(6-hydroxyhexyll)-10-oxatricyclo[5.2.1.0]
decane-3,5-dione (7b) and diphenyl chlorophosphite to yield the
title compound (9b) as a colourless oil (75%).
B
clo[5.2.1.0]decane-3,5-dione (7e) and diethyl chlorophosphate to
yield the title compound (8e) as a clear oil (63%).
1H NMR (CDCl3): d 4.85, dist t, 2H; 4.10, app quintet, J = 7.1 Hz,
4H; 4.04, app quintet, J = 6.8 Hz, 2H; 3.45, t, J = 7.2 Hz, 2H; 2.89, s,
2H; 1.83, m, 2H; 1.63, m, 4H; 1.43, m, 2H; 1.36, m, 4H; 1.34, t,
J = 7.1 Hz, 6H.
1H NMR (CDCl3): d 7.28, m, 5H; 7.15, m, 5H; 4.80, dist t, 2H;
4.20, ABq, J = 6.7 Hz, 1H; 4.16, ABq, J = 6.7 Hz, 1H; 3.39, d,
J = 7.2 Hz, 2H; 2.79, s, 2H; 1.79, m, 2H; 1.55, m, 6H; 1.23, m, 4H.
13C NMR (CDCl3): d 177.1, 150.6, 129.7, 125.2, 120.0, 79.0, 69.1,
49.8, 38.7, 29.8, 28.5, 27.3, 26.0, 24.8 ppm.
13C NMR (CDCl3): d 177.0, 78.7, 67.1, 63.3, 49.5, 38.5, 29.7, 28.3,
27.0, 25.8, 24.6, 15.8 ppm.
MS(ES) m/z: 500 (M+H).
MS(EI) m/z: 402.
HR-MS (ESI, M+H) m/z: calcd for C26H31NO7P: 500.18381; found
500.18389.
4.2.2.1.13. 4-Aza-4-(3-{diphenylphosphate}propyl)-10-oxatricy-
clo[5.2.1.0]decane-3,5-dione (9c). Synthesised as described in
HR-MS (ESI, M+H) m/z: calcd for C18H31NO7P: 404.18381;
found: 404.18389.
4.2.2.1.8.
4-Aza-4-(4-{diethylphosphate}butyl)-10-oxatricy-
clo[5.2.1.0]decane-3,5-dione (8d). Synthesised as described in
Method B from 4-aza-4-(4-hydroxybutyl)-10-oxatricyclo[5.2.1.0]
decane-3,5-dione (7d) and diethyl chlorophosphate to yield the ti-
tle compound (8d) as a pale yellow oil (37%).
1H NMR (CDCl3): d 4.80, br s, 2H; 4.05, q, J = 7.2 Hz, 4H; 3.98, m,
2H; 3.44, dist t, 2H; 2.82, s, 2H; 1.78, m, 2H; 1.56, m, 6H; 1.28, t,
J = 7.2 Hz, 6H.
Method
B
from
4-aza-4-(3-hydroxypropyl)-10-oxatricy-
clo[5.2.1.0]decane-3,5-dione (7c) and diphenyl chlorophosphate
to yield the title compound (9c) as a colourless oil (83%).
1H NMR (CDCl3): d 7.28, m, 3H; 7.16, m, 6H; 4.79 (dd, J = 3.1,
2.2 Hz, 2H), 4.20, dd, J = 13.6, 6.2 Hz, 2H; 3.53, t, J = 7.0 Hz, 2H;
2.79, s, 2H; 1.91, m, 2H; 1.76, m, 2H; 1.50, m, 2H.
13C NMR (CDCl3): d 176.8, 150.2, 129.6, 125.2, 117.8, 79.8, 66.5,
49.6, 35.3, 28.3 ppm.
13C NMR (CDCl3): d 177.1, 79.0, 66.7, 63.7, 49.8, 38.3, 28.5, 27.3,
23.7, 16.1 ppm.
MS(ES) m/z: 458 (M+H).
MS(EI) m/z: 375.
HR-MS (ESI, M+H) m/z: calcd for C16H27NO7P: 376.15251; found
376.15255.
HR-MS (ESI, M+H) m/z: calcd for C23H25NO7P: 458.13686; found
458.13690.
4.2.2.1.14.
4-Aza-4-(4-{diphenylphosphate}butyl)-10-oxatricy-
4.2.2.1.9.
4-Aza-4-(5-{diethylphosphate}pentyl)-10-oxatricy-
clo[5.2.1.0]decane-3,5-dione (9d). Synthesised as described in
Method B from 4-aza-4-(4-hydroxybutyl)-10-oxatricyclo[5.2.1.0]
clo[5.2.1.0]decane-3,5-dione (8e). Synthesised as described in

5740
M. J. Robertson et al. / Bioorg. Med. Chem. 19 (2011) 5734–5741
decane-3,5-dione (7d) and diphenyl chlorophosphate to yield the
title compound (9d) as a colourless oil (70%).
described in Method B from 4-aza-4-(3-hydroxypropyl)-10-
oxatricyclo[5.2.1.0]decane-3,5-dione (7c) and bis(2,2,2-trichlo-
roethyl)chlorophosphate to yield the title compound (10c) as a
colourless oil (71%).
1H NMR (CDCl3): d 7.30, m, 5H; 7.17, m, 5H; 4.80, dist t, 2H;
4.20, dist t, 2H; 3.44, dist t, 2H; 2.80, s, 2H; 1.76, m, 2H; 1.59, m, 6H.
13C NMR (CDCl3): d 177.0, 150.5, 129.7, 125.2, 120.1, 79.0, 68.4,
49.9, 38.1, 28.5, 27.2, 23.6 ppm.
1H NMR (CDCl3): d 4.83, dd, J = 3.1, 2.2 Hz, 2H; 4.62, dABq,
J = 11.2 Hz, 6.6 Hz, 4.14, dt, J = 6.5 Hz, 6.5 Hz, 2H; 3.58, t,
J = 6.7 Hz, 2H; 2.84, s, 2H; 1.97, m, 2H; 1.78, m, 2H; 1.57, m,
2H.
MS(ES) m/z: 472 (M+H).
HR-MS (ESI, M+H) m/z: calcd for C24H27NO7P: 472.15251;
472.15262.
13C NMR (CDCl3): d 176.5, 94.1, 78.5, 76.5, 65.8, 49.3, 34.7, 28.7,
27.4 ppm.
4.2.2.1.15. 4-Aza-4-(5-{diphenylphosphate}pentyl)-10-oxatricy-
clo[5.2.1.0]decane-3,5-dione (9e). Synthesised as described in
Method B from 4-aza-4-(5-hydroxypentyl)-10-oxatricyclo[5.2.1.0]
decane-3,5-dione (7e) and diphenyl chlorophosphite to yield the
title compound (9e) as a pale yellow oil (72%).
1H NMR (CDCl3): d 7.29, m, 5H; 7.14, m, 5H; 4.79, dist t, 2H;
4.19, ABq, J = 6.5 Hz, 1H; 4.15, ABq, J = 6.5 Hz, 1H; 3.38, d,
J = 7.2 Hz, 2H; 2.78, s, 2H; 1.76, m, 2H; 1.66, m, 2H, 1.53, m, 4H;
1.28, m, 2H.
MS(ES) m/z: 566 (M+H).
HR-MS (ESI, M+H) m/z: calcd for C15H19Cl6NO7P: 565.90303;
found 565.90309.
4.2.2.1.20. 4-Aza-4-(4-{bis(2,2,2-trichloroethyl)phosphate}butyl)-
10-oxatricyclo[5.2.1.0]decane-3,5-dione (10d). Synthesised as de-
scribed in Method B from 4-aza-4-(4-hydroxybutyl)-10-oxatricy-
clo[5.2.1.0]decane-3,5-dione (7d) and bis(2,2,2-trichloroethyl)
chlorophosphate to yield the title compound (10d) as a pale yellow
oil (76%).
13C NMR (CDCl3): d 177.0, 150.5, 129.7, 125.2, 120.0, 79.0, 68.9,
49.8, 38.5, 29.5, 28.5, 26.9, 22.4 ppm.
MS(ES) m/z: 486 (M+H).
HR-MS (ESI, M+H) m/z: calcd for C25H29NO7P: 486.16816; found
486.16818.
1H NMR (CDCl3): d 4.77, dist t, 2H; 4.57, d, J = 6.6 Hz, 2H; 4.56, d,
J = 6.6 Hz, 2H; 4.15, dist t, 2H; 3.44, dist t, 2H; 2.81, s, 2H; 1.77, m,
2H; 1.64, m, 4H; 1.56, m, 2H.
13C NMR (CDCl3): d 176.9, 94.6, 94.5, 78.8, 68.4, 49.6, 37.8, 28.3,
26.8, 23.3 ppm.
4.2.2.1.16. 4-Aza-4-(2-{diphenylphosphate}cyclohexyl)-10-oxatri-
cyclo[5.2.1.0]decane-3,5-dione (9f). Synthesised as described in
MS(ES) m/z: 584 (M+H).
Method
B
from 4-aza-4-(2-hydroxycyclohexyll)-10-oxatricy-
HR-MS (ESI, M+H) m/z: calcd for C16H21Cl6NO7P: 579.91868;
found 579.91872.
clo[5.2.1.0]decane-3,5-dione (7f) and diphenyl chlorophosphate
to yield the title compound (9f) as a white solid (70%), mp: 110–
112 °C.
4.2.2.1.21. 4-Aza-4-(5-{bis(2,2,2-trichloroethyl)phosphate}pentyl)-
10-oxatricyclo[5.2.1.0]decane-3,5-dione (10e). Synthesised as de-
scribed in Method B from 4-aza-4-(5-hydroxypentyl)-10-oxatricy-
clo[5.2.1.0]decane-3,5-dione (7e) and bis(2,2,2-trichloroethyl)
chlorophosphate to yield the title compound (10e) as a white solid
(52%), mp: 82–84 °C.
1H NMR (CDCl3): d 7.30, m, 5H; 7.17, m, 5H; 4.79, dist t, 2H;
4.53, m, 1H; 3.90, m, 1H; 2.74, s, 2H; 2.17, m, 4H; 1.78, m, 2H;
1.60, m, 6H.
13C NMR (CDCl3): d 177.1, 150.7, 129.7, 125.2, 120.1, 79.2, 78.0,
50.0, 49.5, 32.4, 28.5, 26.1 ppm.
1H NMR (CDCl3): d 4.79, dist t, 2H; 4.57, d, J = 6.5 Hz, 2H; 4.56, d,
J = 6.5 Hz, 2H; 4.17, ABq, J = 7.1 Hz, 1H; 4.12, ABq, J = 7.1 Hz, 1H;
3.41, d, J = 7.1 Hz, 2H; 2.79, s, 2H; 1.77, m, 2H; 1.70, m, 2H; 1.55,
m, 4H; 1.32, m, 2H.
MS(ES) m/z: 498 (M+H).
HR-MS (ESI, M+H) m/z: calcd for C26H29NO7P: 498.16816; found
498.16820.
4.2.2.1.17. 4-Aza-4-(2-{bis(2,2,2-trichloroethyl)phosphate}ethyl)-
10-oxatricyclo[5.2.1.0]decane-3,5-dione (10a). Synthesised as
described in Method B from 4-aza-4-(2-hydroxyethyl)-10-oxatri-
cyclo[5.2.1.0]decane-3,5-dione (7a) and bis(2,2,2-trichloroethyl)
chlorophosphate to yield the title compound (10a) as a colour-
less oil (60%).
13C NMR (CDCl3): d 177.0, 94.7, 94.6, 78.9, 69.1, 49.8, 38.4, 29.4,
28.5, 26.8, 22.3 ppm.
MS(ES) m/z: 596 (M+H).
HR-MS (ESI, M+H) m/z: calcd for C17H23Cl6NO7P: 593.93433;
found 593.93441.
4.2.2.1.22. 4-Aza-4-(2-{bis(2,2,2-trichloroethyl)phosphate}cyclo-
hexyl)-10-oxatricyclo[5.2.1.0]decane-3,5-dione (10f). Synthesised
as described in Method B from 4-aza-4-(2-hydroxycyclohexyll)-
10-oxatricyclo[5.2.1.0]decane-3,5-dione (7f) and bis(2,2,2-trichlo-
roethyl)chlorophosphate to yield the title compound (10f) as a
white solid (58%), mp: 181–184 °C.
1H NMR (CDCl3): d 5.25, s, 2H; 4.80, dd, J = 3.0, 2.2 Hz, 2H, dd;
4.57, ABq, J = 9.4, 4.7 Hz, 4H; 4.28, ABq, J = 8.8, 5.1 Hz, 2H; 3.75, t,
J = 4.8, 4.8 Hz, 2H; 1.75, m, 2H; 1.55, m, 2H.
13C NMR (CDCl3): 176.4, 78.4, 64.3, 59.7, 49.5, 38.2, 27.9, 13.6.
MS(ES) m/z: 551 (M+H).
HR-MS (ESI, M+H) m/z: calcd for C14H17Cl6NO7P: 551.88738;
found 551.88729.
4.2.2.1.18. 4-Aza-4-(6-{bis(2,2,2-trichloroethyl)phosphate}hexyl)-
10-oxatricyclo[5.2.1.0]decane-3,5-dione (10b). Synthesised as
1H NMR (CDCl3): d 4.83, dist t, 2H; 4.62, d, J = 6.3 Hz, 4H; 4.53,
m, 1H; 3.95, m, 1H; 2.79, s, 2H; 2.26, m, 4H; 1.83, m, 2H; 1.60,
m, 6H.
13C NMR (CDCl3): d 177.1, 94.5, 79.2, 78.4, 49.9, 49.6, 32.5, 28.6,
26.1 ppm.
described in Method
B from 4-aza-4-(6-hydroxyhexyll)-10-
oxatricyclo[5.2.1.0]decane-3,5-dione (7b) and bis(2,2,2-trichlo-
roethyl)chlorophosphate to yield the title compound (10b) as a
pale yellow oil (61%).
MS(ES) m/z: 671 (M+HCOOH+H2O).
HR-MS (ESI, M+H) m/z: calcd for C18H22Cl6NO7P: 604.92650;
found 604.92657.
1H NMR (CDCl3): d 4.84, br s, 2H; 4.63, d, J = 6.3 Hz, 4H; 4.24, ABq,
J = 6.7 Hz, 1H; 4.19, ABq, J = 6.7 Hz, 1H; 3.46, d, J = 7.1 Hz, 2H; 2.86, s,
2H; 1.83, m, 2H; 1.74, m, 2H; 1.56, m, 4H; 1.42, m, 2H; 1.30, m, 2H.
13C NMR (CDCl3): d 177.1, 94.6, 78.9, 69.3, 49.7, 38.6, 29.7, 28.5,
27.1, 25.8, 24.6 ppm.
4.2.2.2. N-(4-Hydroxybutyl)phthalimide (11).
A solution of
phthalic anhydride (50 mg, 3.4 mmol) and 4-aminobutanol
(300 mg, 3.4 mmol) in toluene (5 mL) was heated under micro-
wave irradiation (200 W, 100 °C) for 20 min. After cooling the sol-
vent was removed in vacuo and the resultant residue dissolved in
ethyl acetate (20 mL). After washing with water (2 ꢁ 20 mL) the
organic layer was dried (MgSO4), filtered and evaporated in vacuo
to yield a pale yellow oil that solidified on standing (0.68 g, 92%),
mp: 52–54 °C.
MS(ES) m/z: 610 (M+H).
HR-MS (ESI, M+H) m/z: calcd for C18H25Cl6NO7P: 607.94998;
found 607.95003.
4.2.2.1.19. 4-Aza-4-(3-{bis(2,2,2-trichloroethyl)phosphate}propyl)-
10-oxatricyclo[5.2.1.0]decane-3,5-dione (10c). Synthesised as

M. J. Robertson et al. / Bioorg. Med. Chem. 19 (2011) 5734–5741
5741
1H NMR (CDCl3): d 7.57, m, 1H; 7.51, m, 2H; 3.48, m, 2H; 3.38, s,
OH; 1.58, m, 2H; 1.43, m, 2H.
16. Kok, S. H.; Hong, C. Y.; Kuo, M. Y.; Lee, C. H.; Lee, J. J.; Lou, I. U.; Lee, M. S.; Hsiao,
M.; Lin, S. K. Oral Oncol. 2003, 39, 19.
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2004, 19, 560.
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136.
19. Deng, L.; Shen, L.; Zhang, J.; Yang, B.; He, Q.; Hu, Y. Can. J. Chem. 2007, 85, 938.
20. Hong, C. Y.; Huang, S. C.; Lin, S. K.; Lee, J. J.; Chueh, L. L.; Lee, C. H. K.; Lin, J. H.;
Hsiao, M. Biochem. Biophys. Res. Commun. 2000, 276, 278.
21. Liao, H. F.; Su, S. L.; Chen, Y. J.; Chou, C. H.; Kuo, C. D. Food Chem. Toxicol. 2007,
45, 1678.
22. Cao, J. H.; Xu, B.; Li, M.; Wu, D. Z.; Huang, W.; Cui, J. R. Ai Zheng 2007, 26, 361.
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Zhi 2010, 90, 2145.
24. Li, X. Q.; Shao, S. H.; Fu, G. L.; Han, X. H.; Gao, H. Chin. J. Integr. Med. 2010, 16,
448.
25. Fan, Y. Z.; Zhao, Z. M.; Fu, J. Y.; Chen, C. Q.; Sun, W. Hepatobiliary Pancreat. Dis.
Int. 2010, 9, 414.
13C NMR (CDCl3): d 167.7, 133.2, 131.2, 122.4, 61.0, 37.0, 29.1,
24.4 ppm.
MS(ES) m/z: 220 (M+H).
HR-MS (ESI, M+H) m/z: calcd for C12H13NO3: 219.08954; found:
219.08957.
4.2.2.3. 4-(1,3-Dioxoisoindolin-2-yl)butyl diphenylphosphate
(12).
Synthesised as described in Method B from N-(4-hydrox-
ybutyl)phthalimide (11) and diphenyl chlorophosphate to yield the
title compound (13) as a clear oil (56%).
1H NMR (CDCl3): d 7.82, m, 2H; 7.68, m, 2H; 7.32, m, 5H; 7.16,
m, 5H; 4.27, m, 2H; 3.67, m, 2H; 1.74, m, 4H.
13C NMR (CDCl3): d 168.1, 150.4, 133.8, 131.9, 129.6, 125.1,
123.0, 119.9, 68.4, 37.0, 27.4, 24.5 ppm.
26. Chang, C.; Zhu, Y.; Tang, X.; Tao, W. Mol. Biol. Rep. 2011, 38, 163.
27. Yang, P. Y.; Chen, M. F.; Tsai, C. H.; Hu, D. N.; Chang, F. R.; Wu, Y. C. Toxicol In
Vitro. 2010, 24, 766.
28. Peng, C.; Liu, X.; Liu, E.; Xu, K.; Niu, W.; Chen, R.; Wang, J.; Zhang, Z.; Lin, P.;
Wang, J.; Agrez, M.; Niu, J. Cancer Sci. 2009, 100, 2302.
29. Yang, H.; Guo, W.; Xu, B.; Li, M.; Cui, J. Anticancer Drugs 2007, 18, 1133.
30. Yang, P.-Y.; Chen, M.-F.; Tsai, C.-H.; Hub, D.-N.; Chang, F.-R.; Wua, Y.-C. Toxicol.
In Vitro 2010, 24, 766.
MS(ES) m/z: 452 (M+H).
HR-MS (ESI, M+H) m/z: calcd for C24H22NO6P: 451.11847; found
451.11849.
31. McCluskey, A.; Bowyer, M. C.; Collins, E.; Sim, A. T. R.; Sakoff, J. A.; Baldwin, M.
L. Bioorg. Med. Chem. Lett. 2000, 10, 1687.
Acknowledgement
32. McCluskey, A.; Walkom, C.; Bowyer, M. C.; Ackland, S. P.; Gardiner, E.; Sakoff, J.
A. Bioorg. Med. Chem. Lett. 2001, 11, 2941.
33. Sakoff, J. A.; Ackland, S. P.; Baldwin, M. L.; Keane, M. A.; McCluskey, A. Invest.
New Drugs 2002, 20, 1.
34. McCluskey, A.; Ackland, S. P.; Bowyer, M. C.; Baldwin, M. L.; Garner, J.; Walkom,
C. C.; Sakoff, J. A. Bioorg. Chem. 2003, 31, 66.
35. Hart, M. E.; Chamberlin, A. R.; Walkom, C.; Sakoff, J. A.; McCluskey, A. Bioorg.
Med. Chem. Lett. 2004, 14, 1969.
36. Hill, T. A.; Stewart, S. G.; Gordon, C. P.; Ackland, S. P.; Gilbert, J.; Sauer, B.;
Sakoff, J. A.; McCluskey, A. ChemMedChem 2008, 3, 1878.
37. McCluskey, A.; Keane, M. A.; Mudgee, L.-M.; Sim, A. T. R.; Sakoff, J. A.; Quinn, R.
J. Eur. J. Med. Chem. 2000, 35, 957.
38. Tatlock, J. H.; Linton, A.; Hou, X. J.; Kissinger, C. R.; Pelletier, L. A.; Showalter, R.
E.; Tempczyk, A.; Villafranca, J. E. Bioorg. Med. Chem. Lett. 1997, 7, 1007.
39. Sodeoka, M.; Baba, Y.; Kobayashi, S.; Hirukawa, N. Bioorg. Med. Chem. Lett. 1997,
7, 1833.
40. Laidley, C. W.; Dauben, W. G.; Guo, Z. R.; Lam, J. Y. L.; Casida, J. E. Bioorg. Med.
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We thank Dr. Scott Stewart for the synthesis of 8a.
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