Synthesis and Cytotoxicity of Octahydroepoxyisoindole-7-carboxylic Acids and Norcantharidin–Amide Hybrids as Norcantharidin Analogues

Article abstract of DOI:10.1002/cmdc.201900180

Octahydroepoxyisoindole analogues of norcantharidin were accessed through a Diels–Alder reaction of an amine-substituted furan with maleic anhydride and subsequent reduction of the bicyclo[2.2.1]heptene olefin. Despite retention of the carboxylate and the ether bridgehead known to impart cytotoxic activity to norcantharidin, none of these analogues displayed notable cytotoxicity against the 11 cell lines examined: HT29 (colon), MCF-7 (breast), A2780 (ovarian), H460 (lung), A431 (skin), Du145 (prostate), BE2-C (neuroblastoma), SJ-G2 and U87 (glioblastoma), MIA (pancreatic), and SMA (spontaneous murine astrocytoma). The incorporation of an amino-substituted system post-synthesis of norcantharidin afforded facile access to 14 acid/amide-substituted norcantharidin analogues. Of these, only four displayed sufficient activity at the initial 25 μm compound screening dose to warrant full evaluation of growth inhibition. Common to these analogues was the presence of a 4-biphenyl moiety, and in particular 3-(2-(furan-2-ylmethyl)-3-(4-biphenylamino)-3-oxopropylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (13 c) and 3-(2-(pyrrole-2-ylmethyl)-3-(4-biphenylamino)-3-oxopropylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (24) displayed high levels of cytotoxicity, returning GI₅₀ values of 15 nm (HT29) to 2.9 μm (U87) and 17 nm (SMA) to 2.8 μm (U87), respectively. These are the most cytotoxic norcantharidin analogues reported to date.

Full text of DOI:10.1002/cmdc.201900180

Accepted Article
Title: Synthesis and cytotoxicity of octahydroepoxyisoindole-7-
carboxylic acids and norcantharidin-amide hybrids as
norcantharidin analogues
Authors: Lacey Hizartzidis, Jayne Gilbert, Christopher P Gordon,
Jennette A Sakoff, and Adam McCluskey
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201900180
Link to VoR: http://dx.doi.org/10.1002/cmdc.201900180
A Journal of
www.chemmedchem.org

10.1002/cmdc.201900180
ChemMedChem
FULL PAPER
Synthesis and cytotoxicity of octahydroepoxyisoindole-7-
carboxylic acids and norcantharidin-amide hybrids as
norcantharidin analogues
Lacey Hizartzidis,[a] Jayne Gilbert,[b] Christopher P Gordon,[a,c] Jennette A Sakoff [b] and Adam
McCluskey *[a]
[a]
Dr L Hizartidis, Dr CP Gordon (0000-0001-7583-5609), Prof A McCluskey (0000-0001-7125-863X)
Department: Chemistry, School of Environmental & Life Sciences
Institution: The University of Newcastle
Address 1 University Drive, Callaghan NSW 2308 Australia
E-mail: Adam.McCluskey@newcastle.edu.au
[b]
Dr J Gilbert (0000-0001-5034-386X), Dr JA Sakoff (0000-0002-7009-5792)
Experimental Therapeutics Group, Department of Medical Oncology
Calvary Mater Hospital
Edith Street, Waratah NSW 2298, Australia.
[c]
Present Address: Department: School of Science and Health
Western Sydney University
Locked Bag 1797, Penrith South DC, NSW 2750, Australia
Supporting information for this article is given via a link at the end of the document.
Abstract:
Octahydroepoxyisoindole
analogues
(7a-n)
of
O
O
norcantharidin were accessed through a Diels-Alder reaction of an
amine substituted furan with maleic anhydride and subsequent
reduction of the bicycle[2.2.1]heptane olefin. Despite retention of the
carboxylate and the ether bridgehead known to impart cytotoxic
activity with norcantharidin, none of these analogues displayed
noteworthy cytotoxicity against the 11 cell lines examined herein:
HT29 (colon); MCF-7 (breast); A2780 (ovarian); H460 (lung); A431
(skin); Du145 (prostate); BE2-C (neuroblastoma); SJ-G2 and U87
(glioblastoma); MIA (pancreatic); and SMA (spontaneous murine
astrocytoma). The incorporation of an amino substituted system post
synthesis of norcantharidin afforded facile access to an acid / amide
substituted norcantharidin analogues 13-26. Of these only 13c, 24-
26 displayed sufficient activity at the initial 25 µM compound screening
dose to warrant full growth inhibition evaluation. Common to these
analogues was the presence of a 4-biphenyl moiety, and in particular
3-(2-(furan-2-ylmethyl)-3-(4-biphenylamino)-3-oxopropylcarbamoyl)-
7-oxabicyclo[2.2.1]heptane-2-carboxylic acid 13c and 3-(2-(pyrrole-2-
ylmethyl)-3-(4-biphenylamino)-3-oxopropylcarbamoyl)-7-
O
O
O
O
O
O
1
2
Figure 1. Chemical structures of cantharidin (
analogue norcantharidin ( ).
1) and the demethylated
2
The dried body of the Mylabris beetle which contains
cantharidin, has a long history of use in Chinese traditional
medicine for treatment of dermal conditions and tumours with the
first chemotherapeutic application reported in 1264.4 Cantharidin
and the demethylated analogue norcantharidin (2) are potent
inhibitors of the serine/threonine protein phosphatases, especially
protein phosphatases 1 and 2A (PP1 and PP2A) (Figure 1).5,6 The
interplay between protein kinases, which predominately
phosphorylate serine and threonine residues, and phosphatases,
which remove phosphate moieties, modulates the vast majority of
cellular signal transduction events including neurotransmission,
muscle contraction, glycogen synthesis, T-cell activation, and cell
proliferation.5,6
oxabicyclo[2.2.1]heptane-2-carboxylic acid 24 displayed high levels of
cytotoxicity returning GI50 values of 15 nM (HT29) to 2.9 µM (U87) and
17 nM (SMA) to 2.8 µM (U87) respectively. These represent the most
cytotoxic norcantharidin analogues reported to date.
Nonetheless, clinical development of cantharidin has been
curtailed by its nephrotoxicity, and current use in Western
medicine has been limited to topical applications.7,8 However, the
demethylated norcantharidin (2) displays no such nephrotoxicity
issues.9
Introduction
Rapid access to norcantharidin is afforded via a room
temperature Diels-Alder reaction of furan and maleic anhydride
followed by hydrogenation of the 5,6-olefin. This active anhydride
is then amenable to a range of other synthetic transformations.
Over the past decade, we and others have exploited the facile
access to the 7-oxa-bicyclo[2.2.11]heptane scaffold in drug
discovery programs geared towards developing cytotoxic
agents.10-18 Norcantharidin analogues also show promise against
Plasmodium falciparum,19 and Haemonchus contortus.20
Adult members of the Meloidae family of Coleoptera
(beetles) deter attacks from many predators by discharging
droplets of cantharidin-laden blood reflexively from their hind
leg joints.1,2 These cantharidin (
1) rich secretions are also
used as a copulation gift to protect the fertilised eggs from
predation (Figure 1).3,4
1
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900180
ChemMedChem
FULL PAPER
Members of the norcantharidin class of compounds includes the
O
O
O
R1
N
R1
i
ii
N
O
norcantharidins,21
the
acid
H
benzoyloxymethyl-substituted
amides,22-24 anhydride modified ethers,25,26 norcantharimides,13,27-
3
4a-n
5a-n
iii
29
the
bis-norcantharimides,24,30
the
tetracyclic
norcantharimides,13
norcantharimides.30
along
with
the
tetracyclic-bis-
CO2H
CO2H
O
O
O
O
iv
Having recently developed an expedient flow chemistry
approach to substituted furans, which gave rise to a series of
analogues with moderate to good levels of cytotoxicity across a
panel of cancer cell lines,31 we rationalised that incorporation of
these furans with maleic anhydride might afford access to novel
N
N
R1
R1
7a-n
6a-n
a
c
e
d
b
Cl
Cl
norcantharidin analogues with enhanced cytotoxicity.
outcomes of these investigations are reported herein.
The
O
Cl
g
f
h
i
j
CF3
F
OH
Results and Discussion
m
n
k
l
N
Thus far few modifications of the norcantharidin scaffold have
resulted in retention or enhancement of the cytotoxicity
associated with this family of compounds. It is known that
removal of the 7-O atom and modifications to the 5,6-bridge of the
bicyclo[2.2.1] moiety reduce analogue potency.32,33 Based on our
prior development of norcantharidin analogues we envisaged two
potentially facile routes to modified norcantharidins; Route A
would afford a family of octahydroepoxyisoindole-7-carboxlic
acids (7a-n) (Scheme 1),34 and Route B would result in the
incorporation of furanyl amines into a second series of novel
norcantharidin analogues (13a-c, Scheme 2).35 Route B offered
the possibility of developing chimeric molecules combining
features from our prior reports of cytotoxic norcantharidin
analogues and cytotoxic acrylamide analogues.36-38 We first
O
Scheme 1. Reagents and conditions. Route A: (i) furan-2-carbaldehyde (3),
amine in MeOH [0.05 M], rt, 0.5 h; (ii) imine (4a-n) in MeOH [0.05 M], H-Cube
Pro™, 10% Pd/C, 50 bar H2, 50 °C, with a flow rate of 1.0 mL.min-1; (iii) maleic
anhydride, diethyl ether, rt, 24 h; (iv) hexahydroisoindole-7-carboxylic acid (6a-
n) in MeOH [0.05 M], H-Cube Pro™, 10% Pd/C, 50 bar H2, 50 °C, with a flow
rate of 1.0 mL.min-1.
Accordingly, we turned our attention to the synthesis of a
series of acid-amide based norcantharimides. In designing these
proposed analogues, we were aware of recent efforts that coupled
norcantharidin with cis-platin to produce hybrid drugs with good
efficacy, this suggested that additional norcantharidin-hybrids
may also elicit a favourable cytotoxic response.41 As we have
previously developed a series of highly cytotoxic acrylamides,36-38
we chose to explore the development of norcantharidin-
acrylamide like chimeric compounds. To this end, and with regard
to the side chains installed in the isoindolene analogues above,
we synthesized a library of furan-based cyanoamides (13a-c)
(Scheme 2).
explored
the
synthesis
of
a
focused
library
of
octahydroepoxyisoindole-7-carboxylic acids (7a-n). Synthesis
commenced with treatment of furfural (3) with a range of amines
(see Table 1 for details) to afford imines (4a-n) which were
subjected to H-cube flow reduction using 10%-Pd/C and 50 bar
H2 at a 1.0 mL min-1 flow rate and this effected smooth conversion
to the furanyl amines 5a-n in good to excellent yields. Diels-Alder
addition with maleic anhydride at room temperature gave the
initial Diels-Alder product that underwent an intramolecular amine
attack of the anhydride to afford the hexahydroepoxyisoindole-7-
carboxylic acids 6a-n, which were readily reduced using 10%-
Pd/C and 50 bar H2 at a 1.0 mL min-1 flow rate to give a focused
library of octahydroisoindolene-7-carboxylic acids, 7a-n. Given
our interest in the potential cytotoxicity of norcantharidin
analogues we screened this focused library against our panel of
eleven cancer cell lines: HT29; MCF-7 (breast); A2780 (ovarian);
H460 (lung); A431 (skin); Du145 (prostate); BE2-C
(neuroblastoma); SJ-G2 and U87 (glioblastoma); MIA
(pancreatic); SMA (spontaneous murine astrocytoma).31 In this
instance, no analogue displayed noteworthy cytotoxicity at the
initial 25 µM screening concentration. This suggests that
although the oxctahydroisoindolene-7-carboxylic acids (7a-n) are
structurally related to norcantharidin, and are interesting scaffolds,
they are not promising for the development of cytotoxic agents.
As such no analogue proceeded to a full dose-response
evaluation, as such exploration of this family of compound ceased
at this point, and our attention turned to alternative analogues.
These findings are in keeping with prior reports of a highly
O
O
O
ii
i
R2
NH2
CN
NC
R2
N
H
R2
N
H
O
CN
O
8a-c
9
10a-c
11a-c
iii
O
O
O
iv
N
H
N
H
R2
R2
N
H
O
O
NH2
CO2H
13a-c
O
12a-c
Scheme 2. Reagents and conditions: Route B: (i) MeOH,
°C, 0.5 h; (ii) cyanoamide (10a-c), piperidine (cat.), EtOH, rt, 1 h; (iii)
acrylamide (11a-c) in MeOH [0.05 M], RaNi, 10 bar H2, 50 °C, with a flow
rate of 1.0 mL.min-1; (iv) acetone, rt, 4 h.
µw, 200 W, 120
Three amines were selected in the development of the first
norcantharidin based library, 13a-c: 4-methyoxybenzylamine (8a),
4-methylbenzylamine (8b) and 4-phenylbenzylamine (8c).
Treatment of 8a-c with methyl cyanoacetate under microwave
irradiation afforded cyanoamides (10a-c) in good yields.
Subsequent Knoevenagel condensation with furan gave 11a-c;
selective flow reduction of the olefin and cyano moieties installed
the primary amines in 12a-c,35 which on stirring with
norcantharidin gave the norcantharidin-amide analogues 13a-c.
The 1H and 13C NMR spectra of 13a-c showed clear evidence of
prescriptive
norcantharidin
analogue
cytotoxicity
pharmacophore.32,33,39,40
2
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900180
ChemMedChem
FULL PAPER
diastereomers, but given the early stage of this study, no attempt
was made to separate. We also note that in two separate studies
on norcantharidin analogues, no discernible difference was noted
between purified stereoisomers.42,43 These three compounds,
13a-c, comprise Library B and the outcomes of the initial
cytotoxicity screening against our panel of eleven cell lines at 25
µM compound concentration is presented in Table 1.
Table 1. Evaluation of the cytotoxicity of 7-oxa-bicyclo[2.2.1]heptane derivatives 13a-c (Library B) against a panel of eleven cancer cell lines. Values are the
percentage of growth inhibition at 25 µM drug concentration.
O
O
N
H
N
H
R
O
CO2H
O
R
HT29[a]
14 ± 7
<10
U87[b]
21 ± 4
26 ± 4
MCF-7[c]
33 ± 3
A2780[d]
14 ± 5
<10
H460[e]
<10
A431[f]
<10
Du145[g]
25 ± 4
BE2-C[h]
24 ± 6
SJ-G2[b]
20 ± 4
MIA[i]
11 ± 2
<10
SMA[j]
10 ± 8
<10
O
13a
13b
16 ± 5
<10
<10
17 ± 0.4
16 ± 6
21 ± 7
88 ± 4
81 ± 9
57 ± 2
58 ± 2
67 ± 3
82 ± 1
60 ± 5
56 ± 4
43 ± 10
87 ± 2
71 ± 7
13c
[a] HT29 (colon carcinoma); [b] U87 and SJ-G2 (glioblastoma); [c] MCF-7 (breast carcinoma); [d] A2780 (ovarian carcinoma); [e] H460 (lung carcinoma); [f] A431(skin
carcinoma); [g] Du145 (prostate carcinoma); [h] BE2-C (neuroblastoma); [i] MIA (pancreatic carcinoma); [j] SMA (spontaneous murine astrocytoma).
furan moieties, which were accessed from the corresponding
Of the three analogues in Library B, only biphenyl substituted
furan aldehydes as per Scheme 2. The detail of the additional
13c showed promising levels of cytotoxicity with >80% growth
furan analogues and outcomes of the cytotoxicity screening of
inhibition against HT29, U87 and A431 cell lines and modest
these analogues, Library C 14-19, is presented in Table 2. This
activity (40-80% growth inhibition) against the remaining cell lines.
library spanned modified furans and exemplars of the parent
Based on this promising activity we expanded the scope of the
amines 8a-c.
Table 2. Evaluation of the cytotoxicity of 7-oxa-bicyclo[2.2.1]heptane derivatives 14-19 (Library C) against a panel of eleven cancer cell lines. Values are the
percentage of growth inhibition at 25 µM drug concentration.
O
O
R
N
N
H
O
H
O
CO2
H
R
HT29[a]
U87[b]
MCF-7[c]
A2780[d]
H460[e]
A431[f]
GI50 M)
Du145[g]
BE2-C[h]
SJ-G2[b]
MIA[i]
SMA[j]
(
µ
O
30 ± 7
22 ± 4
<10
29 ± 1
13 ± 4
13 ± 3
18 ± 2
36 ± 16
<10
<10
<10
20 ± 3
14 ± 8
10 ± 11
14
O
12 ± 5
10 ± 3
26 ± 3
30 ± 3
25 ± 4
31 ± 5
<10
<10
22 ± 4
23 ± 2
15 ± 1
12 ± 3
17 ± 6
24 ± 3
<10
<10
<10
OH
15
16
O
<10
11 ± 5
O
O
R
N
N
H
O
H
CO2
H
O
28 ± 2
19 ± 1
1 ± 11
27 ± 2
16 ± 1
32 ± 21
6 ± 5
<10
23 ± 4
11 ± 10
10 ± 9
OH
17
3
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900180
ChemMedChem
FULL PAPER
O
O
R
N
N
H
O
H
CO2
H
O
26 ± 6
21 ± 10
18 ± 5
15 ± 4
23 ± 4
39 ± 2
27 ± 7
19 ± 5
38 ± 19
43 ± 15
9 ± 5
<10
<10
36 ± 5
<10
15 ± 4
10 ± 3
35 ± 19
37 ± 16
OH
18
19
O
36 ± 7
38 ± 6
5 ± 21
[a]
[b]
HT29 (colon carcinoma);
U87 and SJ-G2 (glioblastoma); c MCF-7 (breast carcinoma); [d] A2780 (ovarian carcinoma); [e] H460 (lung carcinoma); [f]
[g]
[h]
[i]
[j]
A431(skin carcinoma);
Du145 (prostate carcinoma);
BE2-C (neuroblastoma); MIA (pancreatic carcinoma); SMA (spontaneous murine
astrocytoma).
As modifications to the furan moiety adversely affected the
As can be seen from the data presented in Table 2, no
potency enhancement was observed, with 14-19 displaying
lower growth inhibition than the lead 13c across all cell lines
evaluated. This was most evident with 18 and 19, based on
the most active parent amine 8c. These data suggest that
additional substituents at the furan moiety are detrimental to
growth inhibition in this panel of cell lines.
observed cytotoxicity we next examined the effect of bioisosteric
modifications of the furan moiety through the synthesis of selected
pyrrole and thiophene analogues. Synthesis was conducted as
per Scheme
2 commencing from pyrrole and thiophene
carboxaldehydes. This gave Library D comprising analogues 20-
26, and the cytotoxicity screening results are shown in Table 3.
Table 3. Evaluation of the cytotoxicity of 7-oxa-bicyclo[2.2.1]heptane derivatives 20-26 (Library D) against a panel of eleven cancer cell lines. Values are the
percentage of growth inhibition at 25 µM drug concentration.
R
HT29[a]
U87[b]
MCF-7[c]
A2780[d]
H460[e]
A431[f]
GI50 M)
Du145[g]
BE2-C[h]
SJ-G2[b]
MIA[i]
SMA[j]
(
µ
HN
<10
27 ± 2
18 ± 5
11 ± 4
15 ± 9
<10
<10
<10
<10
<10
<10
16 ± 2
22 ± 4
<10
<10
20
S
28 ± 7
31 ± 4
18 ± 10
36 ± 18
13 ± 10
10 ± 7
12 ± 12
21
HN
31 ± 6
29 ± 8
27 ± 8
20 ± 5
18 ± 4
<10
28 ± 4
31 ± 3
18 ± 10
20 ± 3
41 ± 15
34 ± 15
<10
<10
<10
20 ± 7
22 ± 2
<10
20 ± 7
10 ± 7
22
S
12 ± 2
12 ± 1
23
86 ± 3
79 ± 2
77 ± 7
74 ± 4
62 ± 3
60 ± 2
57 ± 3
60 ± 1
71 ± 3
57 ± 2
81 ± 2
86 ± 1
65 ± 5
62 ± 2
54 ± 4
77 ± 2
59 ± 4
38 ± 2
87 ± 3
85 ± 1
70 ± 7
69 ± 2
24
25
92 ± 2
>100
54 ± 4
64 ± 6
78 ± 3
47 ± 11
14 ± 5
>100
>100
79 ± 7
73 ± 8
26
[a] HT29 (colon carcinoma); [b] U87 and SJ-G2 (glioblastoma); [c] MCF-7 (breast carcinoma); [d] A2780 (ovarian carcinoma); [e] H460 (lung carcinoma); [f]
[g]
[h]
[i]
[j]
A431(skin carcinoma);
astrocytoma).
Du145 (prostate carcinoma);
BE2-C (neuroblastoma); MIA (pancreatic carcinoma); SMA (spontaneous murine
The pyrrole (20
,
22) and thiophene (21
,
23) analogues
26 based on the biphenyl cyanoamide 10c, returned more
promising levels of cytotoxicity with pyrrole (24) at 54-87%;
thiophene (25) at 38-86%; and indole (26) at 47-100% growth
developed from cyanoamides 10a and 10b, were essentially
inactive across the cell line panel examined. Analogues 24
-
4
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900180
ChemMedChem
FULL PAPER
From the analogues developed herein, the biphenyl
substituted 13c and 24-26 showed sufficient activity to warrant full
dose response evaluation and this data is presented in Table 4.
inhibition. Attempts to enhance the cytotoxicity of thiophene
based analogues through the introduction of
a
4-
triifluoromethyl or
unsuccessful.
a
diphenylmethamine moiety was
Table 4. Evaluation of the cytotoxicity (GI50 (µM)) of 7-oxa-bicyclo[2.2.1]heptane derivatives 13a and 24-26 against a panel of eleven cancer cell lines.
R
HT29[a]
U87[b]
MCF-7[c]
A2780[d]
H460[e]
1.6 ± 0.71
0.2 ± 0.11
12 ± 4.1
A431[f]
GI50 M)
Du145[g]
BE2-C[h]
SJ-G2[b]
MIA[i]
SMA[j]
(
µ
0.015 ±
0.004
0.027 ±
0.001
0.13 ±
0.09
0.045 ±
0.02
0.026 ±
0.003
0.029 ±
0.01
0.056 ±
0.022
0.11 ±
0.091
k
2.9 ± 1.3
2.8 ± 1.6
5.1 ± 1.0
-
13c
24
0.056 ±
0.01
0.036 ±
0.007
0.062 ±
0.02
0.065 ±
0.02
0.029 ±
0.007
0.051 ±
0.01
0.10 ±
0.003
0.017
-
±0.003
0.39 ±
0.11
0.49 ±
0.20
0.79 ±
0.08
4.8 ± 4.1
23 ± 2.3
1.4 ± 0.26
30 ± 0.67
1.0 ± 0.20
17 ± 0.67
31 ± 2.5
1.0 ± 0.14
20 ± 1.2
1.7 ± 0.36
18 ± 3.7
25
15 ±
34 ± 0.58
26 ± 2.0
16 ± 0.33
30 ± 0.33
13 ± 0.88
0.000
26
[a] HT29 (colon carcinoma); [b] U87 and SJ-G2 (glioblastoma); [c] MCF-7 (breast carcinoma); [d] A2780 (ovarian carcinoma); [e] H460 (lung carcinoma); [f] A431(skin
carcinoma); [g] Du145 (prostate carcinoma); [h] BE2-C (neuroblastoma); [i] MIA (pancreatic carcinoma); [j] SMA (spontaneous murine astrocytoma); [k] not determined
Analysis of the data presented in Table 4 reveals that these
norcantharidin analogues display modest (26, 15 – 34 µM) to
O
O
N
excellent levels of cytotoxicity (13c, 24 and 25). Furan (13c) and
O
H
N
H
O
NH
pyrrole (24) are highly cytotoxic with GI50 values ranging from 15
nM (HT29) to 2.9 µM (U87) and 17 nM (SMA) to 2.8 µM (U87)
respectively. Only modest activity was noted against the SJ-G2
cell line. The isosteric manipulation of the furan ‘O’ to the pyrrole
‘NH’ is well tolerated, but the thiophene ‘S’ (25) displayed poorer
activity (>10 fold less active) than the furan and pyrrole analogues
13c and 24. However, the introduction of the additional phenyl
moiety with indole 26 had a detrimental effect on cytotoxicity. This
is consistent with the effects of additional substituents on the furan
core observed earlier (Table 2) suggesting a size restriction in this
region.
CO2H
NH
CO2H
F
28
27
PP2A IC50 = 18.5±3.5 µM
PP1 IC50 = 14.1±7.90 µM
PP2A IC50 = 7.25±1.25 µM
PP1 IC50 = 4.5±1.55 µM
Figure 2. Example norcantharidin analogues 27 and 28 with the PP1 and PP2A
inhibition data shown.
Conclusions
Cantharidin reduces cell viability in a time, concentration and
cell line dependent manner. Clinically cantharidin stimulates the
production of white blood cells by bone marrow contrasting most
other anticancer drugs. It is widely held that cantharidin, and its
analogues, elicit their anticancer effects through the inhibition of
PP2A. Others and we have shown that cantharidin analogues
transiently accelerate cells through the S-phase of the cycle
leading, resulting in cell defects and ultimately cell death. Within
the known series of cantharidin analogues, those possessing at
least one carboxylic acid moiety are known to inhibit both PP1 and
PP2A, and while not the focus of this body of work, we have
previously reported that analogues similar to those described
herein are potent PP1 and PP2A inhibitors, e.g. 27 and 28 (Figure
2). The cytotoxicity data determined with the norcantharidin
analogues reported herein is consistent with the inhibition of
protein phosphatases (but not confirmed by PP1 and PP2A
inhibition studies), and consistent with all other literature reports
to this effect.39,40
Rapid modification of the norcantharidin scaffold is achievable,
but not all modifications give rise to notable levels of cytotoxicity.
The incorporation of furanyl amine moieties prior to the Diels-
Alder addition of maleic anhydride afforded
a family of
octahydroepoxyisoindoles (7a-n) that while retaining the oxa-
bicyclo bridgehead and free carboxylate, known to impart
cytotoxicity in related norcantharidin analogue, the inclusion of the
fused lactam abolished cytotoxicity. However, the ease of
synthetic access to this rigid scaffold may be of interest in other
medicinal chemistry programs. Flow reduction of a family of
cyanoamides to the corresponding amines (11a-c), and their
subsequent reaction with norcantharidin gave rise to a novel
series of norcantharidin analogues.
The initial library
development and screening revealed the presence of a terminal
4-biphenyl moiety (13c) imparted good levels of cytotoxicity, but
that additional substituents on the pendant furan moiety were
detrimental to activity. Isosteric modification of the furan moiety
gave rise to the pyrrole (24), thiophene (25) and indole (26)
analogues, which proceeded to full dose-response evaluation. Of
these four norcantharidin analogues, 13c and 24 showed highly
promising levels of cytotoxicity, typically in the sub 100 nM
potency range except against U87, A2780, H460 and SMA for
5
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900180
ChemMedChem
FULL PAPER
3-Amino-2-(furan-2-ylmethyl)-N-(4-methoxybenzyl)propanamide
(12a)
and norcantharidin (2) (1.4 equivalents) were dissolved separately in
acetone (2 x 10 mL) and the solutions were added together and the
reaction was left to stir at room temperature for 4 hours. The solution was
filtered and washed with cold acetone and dried under suction to afford
13a as a white solid (0.30 g, 78 %); mp 168–170 °C. LRMS (ESI-) m/z 455
[M-H]-. HRMS calc’d for C24H27N2O7, [M-H]- 455.1824; Found 455.1830.
1H NMR (DMSO-d6, 400 MHz): δ 11.91 (br s, OH), 8.21 (t, J = 5.4 Hz, NH),
7.49 (d, J = 1.0 Hz, NH), 7.40 (t, J = 5.9 Hz, NH), 7.07 (d, J = 8.6 Hz, 2H),
6.83 (d, J = 8.6 Hz, 2H), 6.34 (dd, J = 3.0, 1.9 Hz, 1H), 6.08 (d, J = 2.9 Hz,
1H), 4.73 (d, J = 3.7 Hz, 1H), 4.40 (d, J = 3.8 Hz, 1H), 4.22 (dd, J = 14.9,
6.0 Hz, 1H), 4.11 (dd, J = 14.9, 5.6 Hz, 1H), 3.72 (s, 3H), 3.24 – 3.18 (m,
1H), 3.07–2.99 (m, 1H), 2.83 (s, 2H), 2.77–2.68 (m, 3H), 1.57–1.40 (m,
4H). 13C NMR (DMSO-d6, 101 MHz) δ 173.0, 172.7, 171.3, 158.6, 153.7,
141.9, 131.7, 128.9, 114.0, 110.8, 106.6, 79.3, 77.2, 55.5, 53.6, 52.0, 45.2,
42.0, 41.3, 29.2, 28.8, 28.6. IR (cm-1) 3295 (NH), 3091 (OH), 2985, 2937
(CH), 1692 (CO), 1651 (C=C), 1562, 1514 (NH bend), 1302 (CH3), 1247
(CO), 818 (p-Ph).
13c; and U87 and H460 for 24. Current focus is directed towards
isomer isolation which appears less onerous than initially
anticipated. While analogues such as 24 contain 5 chiral centres,
current analysis suggests a degree of stereoselectivity is imparted
throughout the synthetic protocol. For example, RP-HPLC
analysis of a racemic sample of 24 indicated the presence of only
3 diastereomers which are readily separable via preparative
HPLC (Supporting Information). Combined, these represent the
most cytotoxic norcantharidin analogues reported thus far, and
they are promising leads for further development.
Experimental Section
In vitro growth inhibition assay
All test agents were prepared as stock solutions (20 mM) in dimethyl
sulfoxide (DMSO) and stored at -20 °C. All cell lines were cultured in a
humidified atmosphere 5% CO2 at 37 ºC. The cancer cell lines were
maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Trace
Biosciences, Australia) supplemented with 10% foetal bovine serum, 10
mM sodium bicarbonate, penicillin (100 IU/mL), streptomycin (100 µg/mL),
and glutamine (2 mM). Cells in logarithmic growth were transferred to 96-
well plates. Cytotoxicity was determined by plating cells in duplicate in 100
µL medium at a density of 2500-4000 cells/well. On day 0, (24 h after
plating) when the cells were in logarithmic growth, 100 µL medium with or
without the test agent was added to each well. After 72 h drug exposure
growth inhibitory effects were evaluated using the MTT (3- [4,5-
dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay and
absorbance read at 540 nm. Percentage growth inhibition was determined
at a fixed drug concentration of 25 µM. A value of 100% is indicative of
complete 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 values on day 0 and those at the end of drug
exposure.27
3-(2-(Furan-2-ylmethyl)-3-(4-methylbenzylamino)-3-
oxopropylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid
(13b).4
Synthesised as described as for 13a from 3-amino-N-(4-
methylbenzylamino)-2-((5-methylfuran-2-yl)methyl)propanamide
(12b)
and norcantharidin (2) to afford 13b as a white solid (diastereomers
collected in a 1:1 ratio) (0.42 g, overall yield 60%); mp 166–167 °C. LRMS
(ESI-) m/z 439 [M-H]-. HRMS calc’d for C24H27N2O6, [M-H]- 439.1875;
Found 439.1878. 1H NMR (DMSO-d6, 400 MHz): δ 11.92 (br s, OH), 8.37
(t, J = 5.6 Hz, NH), 8.22 (t, J = 5.5 Hz, NH)*, 7.62 (t, J = 5.6 Hz, NH), 7.42
(t, J = 5.4 Hz, NH)*, 7.12–7.01 (m, 4H), 6.34 (s, 1H), 6.10 (dd, J = 12.0,
2.7 Hz, 1H), 4.72 (d, J = 10.0 Hz, 1H), 4.41 (s, 1H), 4.27–4.13 (m, 2H),
3.25–3.21 (m, 2H), 3.06–3.03 (m, 2H), 2.85–2.68 (m, 5H), 2.26 (s, 3H),
1.56–1.40 (m, 4H). 13C NMR (DMSO-d6, 101 MHz,) δ 173.0, 172.9*, 172.9,
172.8*, 171.3, 171.2*, 153.7, 153.7*, 141.9, 141.9*, 136.8, 136.7*, 136.1,
136.0*, 129.2, 127.6*, 110.8, 106.5, 106.6*, 79.3, 79.2*, 77.2, 77.2*, 53.6,
53.3*, 52.0, 51.7*, 45.2, 45.0*, 42.3, 41.3, 31.2, 29.4, 29.2*, 28.9, 28.8*,
28.8, 28.6*, 21.1. IR (cm-1) 3306 (NH), 3001 (OH), 2984, 2947, 2919, 2871
(CH), 1729 (CO), 1692 (CO), 1644 (C=C), 1537 (NH bend), 1381 (CH3),
1251 (C-O), 1180 (C-O), 835 (p-C Ph), 738 (CH2 bend). *Diastereomers
peaks.
All reagents were purchased from Sigma Aldrich and were used without
purification, with the exception of furfural, which was distilled from glass
prior to use. Solvents were bulk, and distilled from glass prior to use.
3-(2-(Furan-2-ylmethyl)-3-(4-biphenylamino)-3-oxopropylcarbamoyl)-
7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (13c).
Synthesised as described as for 13a from 3-amino-N-(4-biphenylamino)-
2-((5-methylfuran-2-yl)methyl)propanamide (12c) and norcantharidin (2)
to afford 13c as a white solid (diastereomers collected in a 1:1 ratio) (0.04
g, overall yield 44%); mp 180 °C. LRMS (ESI-) m/z 501 [M-H]-. HRMS
calc’d for C29H29N2O6, [M-H]- 501.2031; Found 501.2039. 1H NMR
(DMSO-d6, 400 MHz): δ 11.89 (br s, 1H), 8.48 (t, J = 5.8 Hz, 1H), 8.33 (t,
J = 5.7 Hz, 1H), 7.64 (d, J = 8.2 Hz, 2H), 7.57 (d, J = 7.9 Hz, 2H), 7.52 (d,
J = 1.0 Hz, 1H), 7.45 (d, J = 7.7 Hz, 2H), 7.35 (d, J = 7.4 Hz, 1H), 7.25 (t,
J = 8.0 Hz, 2H), 6.39–6.33 (m, 1H), 6.13 (dd, J = 11.2, 2.9 Hz, 1H), 4.73
(d, J = 9.1 Hz, 1H), 4.46–4.42 (m, 1H), 4.34–4.22 (m, 2H), 3.25 (d, J = 6.7
Hz, 1H), 3.09–3.02 (m, 1H), 2.80 (dd, J = 24.0, 14.1 Hz, 5H), 1.56–1.43
(m, 4H). 13C NMR (DMSO-d6, 101 MHz): δ 173.1, 173.0, 171.3, 153.6,
141.9, 140.5, 139.2, 139.0, 129.4, 128.2, 127.8, 127.0, 126.9, 110.8, 106.6,
79.2, 77.3, 53.6, 53.3*, 51.7, 45.2, 45.0*, 42.2, 41.3, 29.4, 28.8*. IR (cm-1)
3306 (NH), 3022 (OH), 2988, 2984, 2915, 2870 (CH), 1698 (CO), 1642
(C=C), 1555, 1490 (NH bend), 1243, 1206 (CO), 1178 (CO), 806 (p-Ph).
*Diastereomers peaks.
1H and 13C NMR spectra were recorded on a Brüker Advance™ AMX 400
MHz spectrometer at 400.1 and 100.1 MHz, respectively. Chemical shifts
(δ) are reported in parts per million (ppm) measured to relative the internal
standards. Coupling constants (J) are expressed in Hertz (Hz). Mass
spectra were recorded on a Shimadzu LCMS 2010 EV using a mobile
phase of 1:1 acetonitirle:H2O with 0.1 % formic acid. Gas chromatography-
mass spectrometry (GC-MS) was performed on a Shimadzu GC-MS
QF2010 EI/NCI System equipped with a ZB-5MS capillary column of 5%
phenylarylene stationary phase.
Melting points were recorded on a BUCHI Melting Point M-565. IR spectra
were recorded on a PerkinElmer Spectrum Two™ FTIR Spectrometer.
Thin layer chromatography (TLC) was performed on Merck 60 F254 pre-
coated aluminium plates with
irradiations were conducted using
microwave, and hydrogenations were performed either using
a
thickness of 0.2 mm. Microwave
a
CEM Discover® Benchmate
a
ThalesNano H-Cube™ or a ThalesNano H-CubeProTM continuous-flow
hydrogenation reactor. All reactions were passed through the H-Cube™
reactor once, unless otherwise specified.
3-[(5-Methylfuran-2-yl)methyl]-3-(4-methylbenzylamino)-3-
oxopropylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid
(14).
Synthesised as described as for 13a from 3-amino-N-(4-
methoxybenzylamino)-2-((5-methylfuran-2-yl)methyl)propanamide (12d)
and norcantharidin (2) to afford 14 as a white solid (diastereomers
collected in a 1:3 ratio) (0.09 g, overall yield 29%); mp 163–165 °C. LRMS
(ESI+) m/z 471 [M+H]+. HRMS calc’d for C25H31N2O7, [M-H]+ 471.2126;
Found 471.2138. 1H NMR (DMSO-d6, 400 MHz): δ11.90 (br s, 1H), 8.34
(t, J = 5.7 Hz, NH)*, 8.19 (t, J = 5.8 Hz, NH), 7.59 (t, J = 5.5 Hz, NH)*, 7.38
Synthesis detail for the precursor compounds 5a-5n, 10a-10e, 11a-11r
and 12a-12r, and final compounds 7a-n is provided in the supporting
material.
3-(2-(Furan-2-ylmethyl)-3-(4-methoxybenzylamino)-3-
oxopropylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid
(13a).4
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900180
ChemMedChem
FULL PAPER
(t, J = 5.8 Hz, NH), 7.09 (dd, J = 8.4, 6.4 Hz, 2H), 6.83 (d, J = 8.6 Hz, 2H),
5.97–5.90 (m, 2H), 4.75–4.69 (m, 1H), 4.40 (d, J = 3.7 Hz, 1H), 4.25–4.08
(m, 2H), 3.72 (s, 3H), 3.25–3.18 (m, 1H), 3.06–2.98 (m, 1H), 2.86–2.61 (m,
5H), 2.18 (s, 3H), 1.59–1.39 (m, 4H). 13C NMR (DMSO-d6, 101 MHz):
173.0, 172.9*, 172.8*, 171.3, 171.2*, 158.6, 151.8, 151.8*, 150.2, 131.9,
131.7*, 128.9, 114.0, 107.2, 106.7, 79.3, 77.2, 55.5, 53.6, 53.3*, 52.0,
51.7*, 45.1, 44.9*, 42.0, 41.3, 29.3, 29.2*, 28.8, 28.7*, 13.7. IR (cm-1) 3285
(NH), 3090 (OH), 2978, 2941 (CH), 1682 (CO), 1648 (C=C), 1562, 1512
(NH bend), 1301 (CH3), 1233 (CO), 816 (p-C Ph). *Diastereomer peaks.
51.6*, 45.0, 44.8*, 42.3, 41.3*, 31.2, 29.4, 29.2*, 28.9, 28.8*, 28.7*, 21.1.
IR (cm-1) 3250 (NH), 3091 (OH), 2987, 2955, 2916 (CH), 1698 (CO), 1645
(C=C), 1566, 1512 (NH bend), 1320 (CH3), 1247 (CO), 808 (p-C Ph).
*Diastereomer peaks.
3-(5-(Hydroxymethyl)furan-2-yl)methyl)-3-(4-biphenylamino)-3-
oxopropylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid
(18).
Synthesised as described as for 14 from 3-amino-N-(4-biphenylamino)-2-
((5-methylfuran-2-yl)hydroxymethyl)propanamide
(12h)
and
3-[(5-(Hydroxymethyl)furan-2-yl)methyl]-3-(4-
methyoxybenzylamino)-3-oxopropylcarbamoyl)-7-
oxabicyclo[2.2.1]heptane-2-carboxylic acid (15).
Synthesised as described as for 14 from 3-amino-2-((5-
(hydroxymethyl)furan-2-yl)methyl)-N-(4-
norcantharidin (2) to afford 18 as a pale brown solid (diastereomers
collected in a 1:1 ratio) (0.10 g, overall yield 38%); mp 151–153 °C. LRMS
(ESI-) m/z 531 [M-H]-. HRMS calc’d for C30H31N2O7, [M-H]- 531.2137;
Found 531.2146. 1H NMR (DMSO-d6, 400 MHz): δ 11.92 (br s, 1H), 8.47
(t, J = 5.9 Hz, 1H), 8.32 (t, J = 5.8 Hz, 1H)*, 7.63 (d, J = 8.0 Hz, 2H), 7.57
(d, J = 7.8 Hz, 2H), 7.45 (d, J = 7.8 Hz, 2H), 7.35 (t, J = 7.3 Hz, 1H), 7.26
(t, J = 7.6 Hz, 2H), 6.16 (d, J = 3.0 Hz, 1H), 6.04 (dd, J = 12.7, 3.0 Hz, 1H),
4.76–4.71 (m, 1H), 4.46–4.41 (m, 1H), 4.36–4.22 (m, 4H), 3.27–3.21 (m,
1H), 3.09 (dd, J = 12.7, 6.5 Hz, 1H), 2.87–2.66 (m, 5H), 1.54–1.39 (m, 4H).
13C NMR (DMSO-d6, 101 MHz): δ 173.1, 173.0*, 172.9, 172.9*, 171.3,
171.2*, 154.4, 152.9, 152.9*, 140.5, 140.5*, 139.2, 139.1, 139.0*, 129.4,
128.2, 127.8, 127.0, 126.9*, 108.0, 107.1, 79.3, 79.3*, 77.2, 77.2*, 56.2,
53.6, 53.3*, 51.9, 51.6*, 45.1, 44.9*, 42.3, 41.4*, 31.2, 29.4, 29.2*, 28.9,
28.8*, 28.8*. IR (cm-1) 3308 (NH), 3021 (OH), 2988, 2947, 2919 (CH), 1722
(CO), 1690 (CO), 1649 (C=C), 1537 (NH bend), 1381 (CH3), 1251 (CO),
1120 (CO), 835 (p-C Ph), 740 (CH2).*Diastereomer peaks.
methoxybenzylamino)propanamide (12e) and norcantharidin (2) to afford
15 as a white solid (diastereomers collected in a 7:3 ratio) (0.10 g, overall
yield 44%); mp 132–136 °C. LRMS (ESI-) m/z 485 [M-H]-. HRMS calc’d
for C25H29N2O8, [M-H]- 485.1929; Found 485.1940. 1H NMR (DMSO-d6,
400 MHz): δ 11.96 (br s, 1H), 8.36 (t, J = 5.8 Hz, NH), 8.21 (t, J = 5.8 Hz,
NH)*, 7.62 (t, J = 5.6 Hz, NH), 7.40 (t, J = 5.8 Hz, NH)*, 7.09 (dd, J = 8.5,
6.6 Hz, 2H), 6.83 (d, J = 8.6 Hz, 2H), 6.13 (d, J = 2.9 Hz, 1H), 6.01 (dd, J
= 13.4, 3.0 Hz, 1H), 5.10 (br s, 1H), 4.72 (dd, J = 12.4, 3.1 Hz, 1H), 4.40
(d, J = 3.6 Hz, 1H), 4.31 (s, 2H), 4.25–4.10 (m, 2H), 3.26–3.21 (m, 1H),
3.08–3.01 (m, 1H), 2.84–2.62 (m, 5H), 2.09 (s, 3H), 1.58–1.40 (m, 4H). 13
C
NMR (DMSO-d6, 101 MHz) δ 173.0, 172.9, 172.7*, 171.3, 158.6, 154.3,
152.9, 152.9*, 131.8, 131.7*, 128.9, 114.0, 108.0, 107.0, 80.1, 79.3, 79.2*,
77.2, 56.1, 55.5, 53.6, 53.3*, 52.0, 51.7*, 51.2*, 45.0, 44.8*, 42.0, 41.3*,
31.2, 29.4, 29.2*, 28.9, 28.7*, 27.9*. IR (cm-1) 3200 (NH), 3056 (OH), 2988,
2949, 2933, 2912 (CH), 1735 (CO), 1641 (C=C), 1565 (NH bend), 1322
(CH3), 1249 (CO), 820 (p-C Ph). *Diastereomer peaks.
3-[2-(Benzofuran-2-yl)methyl]-3-(4-biphenylamino)-3-
oxopropylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid
(19).
Synthesised as described as for 14 from 3-amino-N-(4-biphenylamino)-2-
((5-methylfuran-2-yl)benzofuran)propanamide (12i) and norcantharidin (2)
to afford 19 as pale brown solid (diastereomers collected in a 1:1 ratio)
(0.03 g, overall yield 21%); mp 154–157 °C. LRMS (ESI-) m/z 551 [M-H]-.
HRMS calc’d for C33H31N2O6, [M-H]- 551.2188; Found 551.2196. 1H NMR
(DMSO-d6, 400 MHz): δ 8.57 (t, J = 5.9 Hz 1H), 8.42 (t, J = 5.9 Hz 1H)*,
7.71 (t, J = 5.2 Hz 1H), 7.56 (t, J = 6.9 Hz, 3H), 7.46 (dd, J = 13.9, 7.1 Hz,
3H), 7.36 (t, J = 6.5 Hz, 3H), 7.27–7.18 (m, 2H), 7.12 (dd, J = 11.5, 8.2 Hz,
2H), 6.60 (d, J = 8.9 Hz, 1H), 4.74–4.69 (m, 1H), 4.51–4.43 (m, 1H), 4.42–
4.32 (m, 1H), 4.24–4.11 (m, 1H), 3.13 (d, J = 6.5 Hz, 1H), 3.02–2.79 (m,
6H), 1.60–1.35 (m, 4H).13C NMR (DMSO-d6, 101 MHz): δ 173.1, 173.1*,
172.8, 172.7*, 171.5, 171.4*, 157.4, 154.5, 140.4, 140.4*, 139.1, 138.9,
138.9*, 138.9*, 129.4, 129.0, 128.0, 127.8, 126.9, 126.8*, 123.9, 123.0,
121.0, 111.2, 103.8, 79.6, 79.2*, 79.2*, 77.3, 77.3*, 53.6, 53.4*, 52.2, 51.9*,
49.9, 45.1, 44.9*, 42.2, 41.4*, 29.4, 29.2*, 28.9, 28.8*, 28.4*. IR (cm-1)
3306 (NH), 3020 (OH), 2984, 2947, 2919, 2871 (CH), 1730 (CO), 1680
(C=C), 1537 (NH bend), 1253 (CO), 835 (p-C Ph). *Diastereomer peaks.
3-[2-(Benzofuran-2-yl)methyl]-3-(4-methoxybenzylamino)-3-
oxopropylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid
(16).
Synthesised as described as for 14 from 3-amino-2-(benzofuran-2-
ylmethyl)-N-(4-methoxybenzylamino)propanamide
(12f)
and
norcantharidin (2) to afford 16 as a white solid (diastereomers collected in
a 2:3 ratio) (0.07 g, overall yield 36%); mp 186–188 °C. LRMS (ESI-) m/z
505 [M-H]-. HRMS calc’d for C28H29N2O7, [M-H]- 505.1980; Found
505.1982. 1H NMR (DMSO-d6, 400 MHz): δ 11.91 (br s, 1H), 8.41 (t, J =
5.9 Hz, NH), 8.26 (t, J = 5.9 Hz, NH)*, 7.69 (t, J = 5.8 Hz, 1H), 7.51 (t, J =
4.5 Hz, NH), 7.55–7.51 (m, 1H), 7.25–7.17 (m, 2H), 6.96 (dd, J = 12.4, 8.6
Hz, 2H), 6.64 (dd, J = 8.6, 6.7 Hz, 2H), 6.56 (d, J = 10.5 Hz, 1H), 4.73 (dd,
J = 13.1, 2.8 Hz, 1H), 4.43 (d, J = 4.0 Hz, 1H), 4.30–4.19 (m, 1H), 4.08 (qd,
J = 5.6, 5.2 Hz 1H), 3.67 (d, J = 1.8 Hz, 3H), 3.28 (d, J = 6.3 Hz, 1H), 3.14–
3.05 (m, 1H), 2.99–2.80 (m, 5H), 1.57–1.39 (m, 4H). 13C NMR (DMSO-d6,
101 MHz) δ 173.0, 172.9*, 172.6, 172.5*, 171.4, 171.3*, 158.5, 157.4,
157.3*, 154.5, 131.7, 131.5*, 129.0, 128.7, 123.8, 123.0, 120.9, 113.9,
111.2, 103.7, 79.3, 79.2*, 77.2, 77.2*, 55.4, 53.6, 53.3*, 52.0, 51.7*, 45.0,
44.8*, 41.9, 41.4*, 29.4, 29.3, 29.1*, 28.9, 28.8*. IR (cm-1) 3280 (NH), 3096
(OH), 2987, 2944, 2920 (CH), 1689 (CO), 1644 (C=C), 1560, 1512 (NH
bend), 1312 (CH3), 1255 (CO), 820 (p-C Ph). *Diastereomer peaks.
3-(2-(Pyrrole-2-ylmethyl)-3-(4-methoxybenzylamino)-3-
oxopropylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid
(20).4
2-Cyano-3-(pyrrole-2-yl)-N-(4-methoxybenzyl)acrylamide (11j) (0.14 g,
0.5 mmol) was dissolved in sufficient EtOH:EtOAc (1:1) (10 mL) to form a
0.05 M solution. This solution was hydrogenated using the H-Cube Pro™
with a RaNi catalyst, 70 bar H2 pressure, 70 °C and a flow rate of 1 mL.min-
1. The solvent was removed in vacuo and to afford 2-((1H-pyrrol-2-
yl)methyl)-3-amino-N-(4-methoxybenzyl)propanamide (12j) as a yellow oil
(diastereomers collected in a 1:1 ratio) (0.11 g, 77%). The crude product
and norcantharidin (2) (0.20 g, 1.8 mmol) were dissolved separately in
acetone (2 x 10 mL) and the solutions were added together and the
reaction was left to stir at room temperature for 4 hours. The solution was
filtered and washed with cold acetone and dried under suction to afford 20
as a white solid (0.06 g, overall yield 38%); mp 166–167 °C. LRMS (ESI-)
m/z 454 [M-H]-. HRMS calc’d for C24H28N3O6, [M-H]- 545.1984; Found
545.1991. 1H NMR (DMSO-d6, 400 MHz): δ 11.89 (br s, OH), 10.39 (s,
NH), 8.20 (t, J = 5.5 Hz, NH), 8.07 (t, J = 5.3 Hz, NH)*, 7.50 (t, J = 5.4 Hz,
NH), 7.29 (t, J = 5.3 Hz, NH)*, 7.07 (d, J = 8.4 Hz, 2H), 6.82 (d, J = 8.5 Hz,
2H), 6.55 (s, 2H), 5.90 – 5.75 (m, 2H), 4.71 (s, 1H), 4.39 (s, 1H), 4.23–4.13
(m, 2H), 3.71 (s, 3H), 3.21–3.15 (m, 1H), 3.07 (dd, J = 12.0, 5.7 Hz, 1H),
2.86–2.61 (m, 5H), 1.55–1.39 (m, 4H). 13C NMR (DMSO-d6, 101 MHz): δ
174.0, 173.0, 172.8*, 171.2, 142.1, 142.0*, 140.5, 139.4, 139.1*, 139.0*,
3-[(5-(Hydroxymethyl)furan-2-yl)methyl]-3-(4-methylbenzylamino)-3-
oxopropylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid
(17).
Synthesised as described as for 14 from 3-amino-N-(4-
methylbenzylamino)-2-((5-methylfuran-2-yl)hydroxymethyl)propanamide
(12g) and norcantharidin (2) to afford 17 as
a pale yellow solid
(diastereomers collected in a 1:1 ratio) (0.18 g, overall yield 58%); mp 129–
132 °C. LRMS (ESI+) m/z 471 [M+H]+. HRMS calc’d for C25H29N2O7, [M-
H]- 471.1980; Found 471.1994. 1H NMR (DMSO-d6, 400 MHz): δ 11.90
(br s, 1H), 8.37 (t, J = 5.8 Hz, NH), 8.23 (t, J = 5.8 Hz, NH)*, 7.64 (t, J =
5.7 Hz, NH), 7.42 (t, J = 5.8 Hz, NH)*, 7.07 (q, J = 8.1 Hz, 4H), 6.14 (d, J
= 2.6 Hz, 1H), 6.02 (dd, J = 13.8, 2.9 Hz, 1H), 5.10 (br s, 1H), 4.75–4.70
(m, 1H), 4.41 (d, J = 3.0 Hz, 1H), 4.31 (s, 2H), 4.26–4.14 (m, 2H), 3.26–
3.19 (m, 1H), 3.05 (dt, J = 12.9, 6.4 Hz, 1H), 2.86–2.66 (m, 5H), 2.26 (s,
3H), 1.56–1.41 (m, 4H). 13C NMR (DMSO-d6, 101 MHz): 173.0, 172.9*,
172.8*, 171.3, 171.2*, 154.3, 152.9, 152.9*, 136.8, 136.7*, 136.1, 136.1*,
129.2, 127.6, 107.9, 107.0, 79.3, 79.2*, 77.2, 77.2*, 56.1, 53.6, 53.3*, 51.9,
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900180
ChemMedChem
FULL PAPER
129.4, 128.4, 128.3, 128.2*, 127.8, 127.2, 127.0, 126.9*, 126.0, 124.5,
79.3, 79.2*, 77.2, 53.7, 53.3*, 51.8, 48.4, 48.1*, 42.2, 41.4*, 31.7, 30.4,
29.4, 29.2, 28.9*, 26.8, 22.5, 14.3. IR (cm-1) 3432 (NH), 3306 (NH), 3074
(OH), 2992, 2937, 2882, 2834 (CH), 1685 (CO), 1653 (CO), 1635 (C=C),
1538, 1513 (NH bend), 1302 (CH3), 1245, 1223 (C-O), 806 (p-C Ph).
*Diastereomer peaks.
3-(2-(Pyrrole-2-ylmethyl)-3-(4-biphenylamino)-3-
oxopropylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid
(24).
Synthesised as described as for 12a from 3-amino-N-(4-biphenylamino)-
2-((5-methylpyrrole-2-yl)methyl)propanamide (12n) and norcantharidin (2)
to afford 24 as a white solid (diastereomers collected in a 7:3 ratio) (0.04
g, 38%); mp 194 °C. LRMS (ESI-) m/z 500 [M-H]-. HRMS calc’d for
C29H30N3O5, [M-H]- 500.2191; Found 500.2200. 1H NMR (DMSO-d6, 400
MHz): δ 11.85 (s, 1H), 10.42 (s, 1H), 8.33 (t, J = 5.8 Hz, 1H), 8.19 (t, J =
5.9 Hz, 1H), 7.63 (d, J = 7.4 Hz, 2H), 7.56 (d, J = 8.1 Hz, 2H), 7.46 (t, J =
7.6 Hz, 2H), 7.35 (t, J = 7.3 Hz, 1H), 7.23 (t, J = 7.3 Hz, 2H), 6.57 (d, J =
1.4 Hz, 1H), 5.90 (dd, J = 5.3, 2.6 Hz, 1H), 5.79 (d, J = 8.6 Hz, 1H), 4.73
(dd, J = 11.3, 2.5 Hz, 1H), 4.44 (s, 1H), 4.36–4.22 (m, 2H), 3.25–3.20 (m,
1H), 3.12–3.03 (m, 1H), 2.87–2.60 (m, 5H), 1.54–1.39 (m, 4H). 13C NMR
(DMSO-d6, 101 MHz): δ 172.6, 172.5*, 171.9, 170.2, 170.1*, 139.4, 138.2,
138.0*, 137.9, 128.4, 128.3, 127.2, 126.7, 125.9, 125.8, 115.4, 106.6,
104.6, 78.2, 76.2, 52.3, 50.9, 50.7*, 45.5, 41.2, 40.3, 28.3, 27.8*, 27.4. IR
(cm-1) 3303 (NH), 3022 (OH), 2991, 2960, 2918, 2855 (CH), 1712 (CO),
1686 (C=C), 1533, 1484 (NH bend), 1242, 1212 (C-O), 1165 (C-O), 801
(p-Ph). *Diastereomer peaks.
3-(2-(Thiophene-2-ylmethyl)-3-(4-methoxybenzylamino)-3-
oxopropylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid
(21).4
Synthesised as described as for 20 from 3-amino-N-(4-
methoxybenzylamino)-2-((5-methylthiophene-2-yl)methyl)propanamide
(12k) and norcantharidin (2) to afford 21 as a white solid (diastereomers
collected in a 1:1 ratio) (0.1 g, overall yield 41%); mp 158–159 °C. LRMS
(ESI-) m/z 471 [M-H]-. HRMS calc’d for C24H27N2O6S, [M-H]- 471.1595;
Found 471.1608. 1H NMR (DMSO-d6, 400 MHz): δ 11.89 (br s, 1H), 8.33
(t, J = 4.7 Hz, NH), 8.20 (t, J = 5.6 Hz, NH)*, 7.58 (t, J = 5.9 Hz, NH), 7.40
(t, J = 6.2 Hz, NH)*, 7.30 (d, J = 5.2 Hz, 1H), 7.25 (t, J = 6.0 Hz, 1H)*, 7.17
(d, J = 8.5 Hz, 2H), 7.03 (t, J = 8.2 Hz, 2H), 6.98–6.87 (m, 1H), 6.90–6.74
(m, 3H), 4.72 (d, J = 9.9 Hz, 1H), 4.41 (d, J = 2.8 Hz, 1H)*, 4.37 (d, J = 4.0
Hz, 1H)*, 4.23–4.09 (m, 2H), 3.71 (s, 3H), 3.26–3.17 (m, 1H), 3.05 (dd, J
= 12.8, 7.5 Hz, 1H), 3.01–2.93 (m, 1H), 2.93–2.66 (m, 4H), 1.55–1.38 (m,
4H). 13C NMR (DMSO-d6, 101 MHz): 172.9, 172.8, 171.2, 158.5, 142.1,
142.0*, 131.8, 129.0*, 128.9, 127.2, 126.0, 124.4, 114.0, 79.2, 77.3, 55.5,
53.4, 48.0, 42.0, 41.3, 30.4, 29.4, 28.9, 22.5. IR (cm-1) 3310 (NH), 3062
(OH), 2986, 2963, 2920, 2874 (CH), 1695 (CO), 1646 (C=C), 1545, (NH
bend), 1382 (CH3), 1244 (CO), 816 (p-C Ph). * Diastereomer peaks.
3-(2-(Thiophene-2-yl)methyl)-3-(4-biphenylamino)-3-
oxopropylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid
(25).4
Synthesised as described as for 20 from 3-amino-N-(4-biphenylamino)-2-
((5-methylthiophene-2-yl)methyl)propanamide (12o) and norcantharidin
(2) to afford 25 as a white solid (diastereomers collected in a 1:1 ratio)
(0.23g, overall yield 46%); mp 171–174 °C. LRMS (ESI-) m/z 471 [M-H]-.
HRMS calc’d for C29H29N2O5S, [M-H]- 471.1803; Found 471.1805. 1H
NMR (DMSO-d6, 400 MHz): δ 11.89 (br s, OH), 8.44 (t, J = 5.3 Hz, NH),
8.31 (t, J = 5.6 Hz, NH)*, 7.63 (d, J = 7.9 Hz, 2H), 7.61 (d, J = 2.1 Hz, 1H)*,
7.59 (d, J = 2.3 Hz, 1H), 7.54 (d, J = 8.2 Hz, 2H), 7.46 (d, J = 7.6 Hz, 2H),
7.35 (dd, J = 8.8, 6.2 Hz, 2H), 7.18 (t, J = 8.6 Hz, 2H), 6.97–6.91 (m, 1H),
6.85 (dd, J = 8.7, 3.2 Hz, 1H), 4.73 (d, J = 11.1 Hz, 1H), 4.50–4.40 (m, 1H),
4.37–4.09 (m, 2H). 3.25 (d, J = 7.0 Hz, 1H), 3.08 (dd, J = 8.0, 5.2 Hz, 1H),
2.96–2.71 (m, 5H), 1.46–1.37 (m, 2H), 1.27–1.17 (m, 2H). 13C NMR
(DMSO-d6, 101 MHz): δ 174.0, 173.0, 172.8, 171.2, 142.1, 142.02, 140.5,
139.4, 139.1, 139.0, 129.4, 128.4, 128.3, 128.2, 127.8, 127.2, 127.0, 126.9,
126.0, 124.5, 79.3, 79.2, 77.2, 53.7, 53.3, 51.8, 48.4, 48.1, 42.2, 41.4, 31.7,
30.4, 29.4, 29.2, 28.9, 26.8, 22.5, 14.3. IR (cm-1) 3316 (NH), 3070 (OH),
3033, 2985, 2923, 2876 (CH), 1691 (CO), 1646 (C=C), 1534, 1487 (NH
bend), 1242 (CO), 819 (p-C Ph). *Diastereomer peaks.
3-(2-(Pyrrole-2-ylmethyl)-3-(4-methylbenzylamino)-3-
oxopropylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid
(22).
Synthesised as described as for 20 from 3-amino-N-(4-
methylbenzylamino)-2-((5-methylpyrrole-2-yl)methyl)propanamide (12l)
and norcantharidin (2) to afford 22 as a pale brown solid (diastereomers
collected in a 1:1 ratio) (0.02 g, overall yield 21%); mp 173–176 °C. LRMS
(ESI-) m/z 438 [M-H]-. HRMS calc’d for C24H28N3O5, [M-H]- 438.2034;
Found 438.2040. 1H NMR (DMSO-d6, 400 MHz): δ 10.39 (s, 1H), 8.23 (t,
J = 5.9 Hz, 1H), 8.10 (t, J = 5.7 Hz, 1H), 7.53 (t, J = 5.8 Hz, 1H), 7.32 (t, J
= 5.9 Hz, 1H)*, 7.09–7.01 (m, 4H), 6.56 (d, J = 1.5 Hz, 1H), 5.89 (dd, J =
5.3, 2.6 Hz, 1H), 5.77 (d, J = 9.0 Hz, 1H), 4.76–4.71 (m, 1H), 4.67 (d, J =
2.6 Hz, 1H)*, 4.44–4.39 (m, 1H), 4.28–4.16 (m, 2H), 3.23–3.18 (m, 1H),
3.10–3.02 (m, 1H), 2.81 (ddd, J = 24.3, 22.1, 12.4 Hz, 5H), 2.26 (s, 3H),
1.57–1.45 (m, 4H). 13C NMR (DMSO-d6, 101 MHz): δ 173.5, 173.4*, 173.0,
171.3, 171.2*, 136.9, 136.7*, 136.1, 136.0*, 129.5, 129.2, 127.6, 116.4,
107.7, 105.7, 79.3, 79.2*, 77.2, 77.2*, 53.7, 53.4*, 52.2, 52.0*, 51.7*, 46.7,
46.5*, 42.3, 41.3, 31.2, 29.3, 29.2*, 29.1, 28.9*, 28.8, 28.5*, 28.4*, 21.1.
IR (cm-1) 3304 (NH), 3031 (OH), 2986, 2967, 2920 (CH), 1735 (CO), 1691
(CO), 1649 (C=C), 1540 (NH bend), 1388 (CH3), 1252 (CO), 1145 (CO),
835 (p-Ph). *Diastereomer peaks.
3-[2-(1H-indol-3-yl)methyl]-3-(4-biphenylamino)-3-
oxopropylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid
(26).
Synthesised as described as for 20, using EtOH:DMF (8:2) as the solvent
for the reduction, from 3-amino-N-(4-biphenylamino)-2-((5-methylpyrrole-
2-yl)1H-indol-3-yl)propanamide (12p) and norcantharidin to afford 26 as a
white solid (diastereomers collected in a 2:1 ratio) (0.02 g, 23%); mp 160–
161 °C. LRMS (ESI-) m/z 550 [M-H]-. HRMS calc’d for C33H32N3O5, [M-
H]- 550.2347; Found 550.2355. 1H NMR (DMSO-d6, 400 MHz): δ 10.83 (s,
1H), 8.39 (t, J = 6.0 Hz, 0.3H)*, 8.25 (t, J = 6.0 Hz, 0.7H)*, 7.62 (d, J = 7.5
Hz, 2H), 7.57 – 7.54 (m, 1H), 7.51 – 7.44 (m, 4H), 7.41 – 7.34 (m, 3H),
7.14 – 7.04 (m, 4H), 6.97 (t, J = 7.4 Hz, 1H), 4.74 – 4.72 (m, 1H), 4.43 –
4.42 (s, 1H), 4.36 – 4.15 (m, 2H), 3.32 – 3.29 (m, 1H, obscured), 3.14 –
3.09 (m, 1H), 2.94 – 2.77 (m, 5H), 1.54 – 1.39 (m, 4H). 13C NMR (DMSO-
d6, 101 MHz): δ 173.6*, 173.5, 172.54, 172.48*, 170.8, 170.7*, 140.04*,
140.02, 138.8*, 138.7, 138.42, 138.39*, 136.2, 128.9, 127.6,
127.34*,127.31, 127.26, 126.5, 126.4, 123.1, 120.8, 118.6*, 118.5, 118.1,
111.82, 111.80*, 111.23, 111.21*, 78.9, 76.8, 53.3, 52.9*, 51.7, 51.4*, 46.7,
46.5*, 41.7, 41.2*, 41.1, 28.9*, 28.7, 28.44*, 28.38, 25.9*, 25.7 IR (cm-1)
3350 (NH), 3012 (OH), 2990, 2956, 2921, 2843 (CH), 1699 (CO), 1653
(C=C), 1579, 1495 (NH bend), 1236, 1199 (C-O), 1168 (C-O), 800 (p-Ph).
*Diastereomer peaks.
3-(2-(Thiophene-2-ylmethyl)-3-(4-methylbenzylamino)-3-
oxopropylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid
(23).4
Synthesised as described as for 20 from 3-amino-N-(4-
methylbenzylamino)-2-((5-methylthiophene-2-yl)methyl)propanamide
(12m) and norcantharidin (2) to afford 23 as a white solid (diastereomers
collected in a 1:1 ratio) (0.13 g, overall yield 38%); mp 161–163 °C. LRMS
(ESI-) m/z 455 [M-H]-. HRMS calc’d for C24H27N2O5S, [M-H]- 455.1646;
Found 455.1650. 1H NMR (DMSO-d6, 400 MHz): δ 11.91 (br s, OH), 8.33
(t, J = 5.8 Hz, NH)*, 8.2 (t, J = 5.8 Hz, NH), 7.61 (t, J = 5.7 Hz, NH)*, 7.41
(t, J = 5.8 Hz, NH), 7.32 (dd, J = 5.1, 0.8 Hz, 1H), 7.05 (d, J = 7.9 Hz, 2H),
6.98 (t, J = 8.5 Hz, 2H), 6.94–6.90 (m, 1H), 6.83 (dd, J = 8.9, 2.9 Hz, 1H),
4.75–4.69 (m, 1H)*, 4.42 (d, J = 3.3 Hz, 1H), 4.27–4.11 (m, 2H), 3.27–3.19
(m, 1H), 3.09–2.95 (m, 2H), 2.89 (dd, J = 12.3, 6.9 Hz, 1H), 2.84–2.67 (m,
2H), 2.51–2.49 (m, 2H), 2.25 (s, 3H), 1.57–1.40 (m, 4H). 13C NMR (DMSO-
d6, 101 MHz): δ 172.9, 172.8*, 172.7, 171.2*, 171.1, 142.1, 142.0*, 136.8,
136.6*, 136.1, 136.0*, 129.1, 127.6, 127.2*, 126.0, 124.4, 80.1, 79.3, 79.2*,
77.2, 77.2*, 53.6, 53.3*, 51.9, 51.7*, 51.2, 48.3, 48.0*, 42.3, 41.3*, 30.4,
30.2*, 29.4, 29.2*, 28.9, 28.8*, 21.1. IR (cm-1) 3310 (NH), 3062 (OH), 2986,
2963, 2920, 2874 (CH), 1695 (CO), 1646 (C=C), 1545, (NH bend), 1382
(CH3), 1244 (CO), 816 (p-C Ph). *Diastereomer peaks.
3-[2-(Thiophen-2-yl)methyl]-3-(4-(trifluoromethyl)benzyl)amino)-3-
oxopropylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid
(27).
Synthesised as described as for 20 from 3-amino-N-(4-
(trifluoromethyl)benzyl
amino)-2-((5-methylthiophene-2-
8
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900180
ChemMedChem
FULL PAPER
1166–1176.
yl)methyl)propanamide (12q) and norcantharidin (2) to afford 27 as a white
solid (diastereomers collected in a 1:1 ratio) (0.12 g, 57%); mp 190–193 °C.
LRMS (ESI-) m/z 509 [M-H]-. HRMS calc’d for C24H25F3N2O5S, [M-H]-
509.1364; Found 509.1369. 1H NMR (DMSO-d6, 400 MHz): δ 11.89 (s,
OH), 8.52 (t, J = 5.8 Hz, NH), 8.38 (t, J = 5.7 Hz, NH)*, 7.67 (t, J = 5.8 Hz,
NH), 7.59 (d, J = 8.1 Hz, NH), 7.46 (t, J = 6.1 Hz, NH)*, 7.33 (dd, J = 5.1,
1.1 Hz, 1H), 7.27 (dd, J = 11.0, 8.4 Hz, 2H), 6.97–6.90 (m, 1H), 6.83 (dd,
J = 8.5, 3.0 Hz, 1H), 4.73 (dd, J = 11.0, 3.1 Hz, 1H), 4.43 (s, 1H), 4.37–
4.25 (m, 2H), 3.28–3.23 (m, 1H), 3.11–3.04 (m, 1H), 3.03–2.79 (m, 5H),
1.58–1.41 (m, 4H). 13C NMR (DMSO-d6, 101 MHz): δ 173.1, 172.9, 171.3,
171.2*, 142.0, 142.0, 128.4, 128.2, 128.2*, 127.2, 126.1, 125.4, 125.4*,
124.5, 79.8, 79.3, 79.2*, 77.3, 53.7, 53.3*, 52.0, 51.7*, 48.4, 48.2*, 42.2,
41.4, 30.4, 29.3, 29.2, 28.9*. IR (cm-1) 3301 (NH), 3056 (OH), 2988, 2975,
2903, 2892 (CH), 1734 (CO), 1642 (C=C), 1524 (NH bend), 1246 (CO),
1170 (CO), 804 (p-C Ph). *Diastereomer peaks.
[14] J. A. Sakoff, S. P. Ackland, M. L. Baldwin, M. A. Keane and A.
McCluskey, Invest. New Drugs, 2002, 20, 1–11.
[15] J. A. Sakoff and A. McCluskey, Curr. Pharm. Des., 2004, 10, 1–21.
[16] A. McCluskey, A. T. R. Sim and J. A. Sakoff, J. Med. Chem., 2002, 45,
1151–1175.
[17] L. H. Zheng, Y. L. Bao, Y. Wu, C. L. Yu, X. Meng and Y. X. Li, Can.
Lett., 2008, 272, 102–109.
[18] C.-H. Hsieh, K. S. C. Chao, H.-F. Liao and Y.-J. Chen, Evid. Based
Compl. Alt. Med., 2013, 2013, article ID 838651, 11 pages.
[19] J. Bajsa, A. McCluskey, C. P. Gordon, S. G. Stewart, T. A. Hill, R.
Sahu, S. O. Duke and B. L. Tekwani, Bioorg. Med. Chem. Lett., 2010,
20, 6688–6695.
[20] B. E. Campbell, M. Tarleton, C. P. Gordon, J. A. Sakoff, J. Gilbert, A.
McCluskey and R. B. Gasser, Bioorg. Med. Chem. Lett., 2011, 21,
3277–3281.
[21] Y. Baba, N. Hirukawa, N. Tanohira and M. Sodeoka, J. Am. Chem.
Soc., 2003, 125, 9740–9749.
[22] C. E. Puerto Galvis, L. Y. Vargas Méndez and V. V. Kouznetsov,
Chem. Biol. Drug Des., 2013, 82, 477-499.
3-[2-(Thiophen-2-yl)methyl]-3-((2,2-diphenylethyl)amino)-3-
oxopropylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid
(28).
[23] T. A. Hill, S. G. Stewart, C. P. Gordon, S. P. Ackland, J. Gilbert, B.
Sauer, J. A. Sakoff and A. McCluskey, ChemMedChem, 2008, 3, 1878–
1892.
Synthesised as described as for 20 from 3-amino-N-(2,2-
diphenylethyl)amino)-2-((5-methylthiophene-2-yl)methyl)propanamide
(12r) and norcantharidin (2) to afford 28 as a white solid (0.05g, 26%); mp
162–164 °C. LRMS (ESI-) m/z 531 [M-H]-. HRMS calc’d for C30H31N2O5S,
[M-H]- 531.1959; Found 531.1969. 1H NMR (DMSO-d6, 400 MHz): δ 11.85
(s, 1H), 8.00 (t, J = 5.2 Hz, NH), 7.40 (t, J = 5.4 Hz, NH), 7.29–7.15 (m,
11H), 6.85 (dd, J = 4.9, 3.5 Hz, 1H), 6.71 (d, J = 2.7 Hz, 1H), 4.71 (d, J =
3.4 Hz, 1H), 4.44 (d, J = 4.0 Hz, 1H), 4.16 (t, J = 7.7 Hz, 1H), 3.73 (dd, J =
13.5, 7.2 Hz, 1H), 3.64 (dd, J = 12.9, 5.6 Hz, 1H), 3.12 (dd, J = 12.7, 6.3
Hz, 1H), 2.92 (dd, J = 13.3, 5.9 Hz, 1H), 2.86–2.71 (m, 4H), 1.56–1.39 (m,
4H). 13C NMR (DMSO-d6, 101 MHz): δ 173.1, 173.0, 171.0, 143.5, 142.1,
128.8, 128.3, 127.2, 126.7, 125.8, 124.3, 79.0, 77.3, 53.4, 51.9, 50.5, 47.7,
43.8, 41.0, 30.1, 29.3, 29.0. IR (cm-1) 3306 (NH), 3012 (OH), 2998, 2985,
2965, 2918, 2884, 2834 (CH), 1712 (CO), 1655 (C=C), 1564 (NH bend),
1243 (CO), 1175 (CO), 800 (p-C Ph).
[24] S. G. Stewart, T. A. Hill, J. Gilbert, S. P. Ackland, J. A. Sakoff and A.
McCluskey, Bioorg. Med. Chem., 2007, 15, 7301–7310.
[25] M. Tarleton, J. Gilbert, J. A. Sakoff and A. McCluskey, Eur. J. Med.
Chem., 2012, 54, 573–581.
[26] B. Sauer, J. Gilbert, J. A. Sakoff and A. McCluskey, Lett. Drug Des.
Disc., 2009, 6, 1–7.
[27] M. J. Robertson, C. P. Gordon, J. Gilbert, A. McCluskey and J. A.
Sakoff, Bioorg. Med. Chem., 2011, 19, 5734–5741.
[28] L. Deng and S. Tang, Expert Opin. Ther. Patents, 2011, 21, 1743–
1753.
[29] T. A. Hill, S. G. Stewart, S. P. Ackland, J. Gilbert, B. Sauer, J. A. Sakoff
and A. McCluskey, Bioorg. Med. Chem., 2007, 15, 6126–6134.
[30] L. Deng, Z. Yong, W. Tao, J. Shen and W. Wang, J. Heterocyclic.
Chem., 2010, 48, 158–161.
[31] J. A. Sakoff and S. P. Ackland, Can. Chemother. Pharmacol., 2000, 46,
477–487.
[32] A. McCluskey, C. Taylor, R. J. Quinn, M. Suganuma, H. Fujiki. Bioorg.
Med. Chem. Lett., 1996, 6, 1025-1028.
[33] A. Thaqi, J. L. Scott, J. Glibert, J. A. Sakoff, A. McCluskey. Eur. J.
Med. Chem., 2010, 45, 1717-1723
[34] C. P. Gordon, N. Byrne and A. McCluskey, Green Chem., 2010, 12,
1000-1006.
Acknowledgements
[35] L. Hizartzidis, M. Tarleton, C. P. Gordon and A. McCluskey, RSC Adv.,
2014, 4, 9709–9722.
[36] M. Tarleton, L. Dyson, J. Gilbert, J. A. Sakoff and A. McCluskey,
This project was supported by the Australian Cancer Research
Foundation, Ramaciotti Foundation and the Australian Research
Bioorg. Med. Chem., 2013, 21, 333–347.
[37] M. Tarleton, J. Gilbert, J. A. Sakoff and A. McCluskey, Eur. J. Med.
Chem., 2012, 57, 65–73.
[38] M. Tarleton, J. Gilbert, M. J. Robertson, A. McCluskey and J. A. Sakoff,
Med. Chem. Commun., 2011, 2, 31-37.
[39] L. Deng, J. Dong, W. Wang. Mini Rev. Med. Chem. 2013, 13, 1166-
1176
Council. LH acknowledges the receipt of
Newcastle Postgraduate Scholarship.
a University of
Keywords: anti-tumour agent • norcantharidin chimera •
structure-activity relationship • Knoevenagel condensation •
growth inhibition
[40] C. E. P. Galvis, L. Y. V. Mendez, V. V. Kouznetsov. Chem. Biol. Drug.
Des. 2013, 82, 477-499.
[41] K. K. W. To, Y.-P. Ho and S. C. F. Au-Yeung, Can. Lett., 2005, 223,
227–237.
[42] A. McCluskey, S. P. Ackland, M. C. Bowyer, M. L. Baldwin, J. Garner,
C. C. Walkom and J. A. Sakoff, Bioorg. Chem., 2003, 31, 68–79.
References:
[43] T. Shimizu, M. Iizuka, H. Matsukura, D. Hashizume and M. Sodeoka,
Chem., Asian J., 2012, 7, 1221–1230.
[1]
M. R. Nikbakhtzadeh, K. Dettner, W. Boland, G. Gäde and S. Dötterl, J.
Insect Physiol., 2007, 53, 890–899.
[2]
[3]
L. Moed, T. A. Shwayder and M. W. Chang, Arch. Derm., 2001, 137,
1357–1360.
T. Eisner, S. R. Smedley, D. K. Young, M. Eisner and B. Roach, Proc.
Natl. Acad. Sci. U.S.A., 1996, 93, 6499–6503.
[4]
[5]
[6]
[7]
G.-S. Wang, J. Enthnopharm., 1989, 26, 147–162.
S. S. Taylor and A. P. Kornev, Trends Biochem. Sci., 2011, 36, 65-77.
Y. Shi, Cell, 2009, 139, 468–484.
R. S. de Jong, E. G. E. de Vries, S. Meijer, P. E. de Jong and N. H.
Mulder, Cancer Chemother. Pharmacol., 1998, 42, 160–164.
F. Massicot, H. Dutertre-Catella, C. Pham-Huy, X.-H. Liu, H. T. Duc and
J.-M. Warnet, Basic Clin Pharmacol Toxicol, 2005, 96, 26–32.
F. Massicot, H. Dutertre-Catella, C. Pham-Huy, X.-H. Liu, H. T. Duc and
J.-M. Warnet, Basic Clin Pharmacol Toxicol, 2005, 96, 26–32.
[8]
[9]
[10] J. H. Cao, B. Xu, D. Z. Wu, W. Huang and J. R. Cui, Chin. J. Can.,
2007, 26, 361–366.
[11] D. Liu and Z. Chen, Antican. Agents. Med. Chem 2009, 9, 392-396.
[12] L. P. Deng, J. Dong, H. Cai and W. Wang, Curr. Med. Chem., 2013, 20,
159–166.
[13] L. Deng, J. Dong and W. Wang, Mini. Rev. Med. Chem., 2013, 13,
9
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900180
ChemMedChem
FULL PAPER
Entry for the Table of Contents
Insert graphic for Table of Contents here. ((Please ensure your graphic is in one of following formats))
O
X = O; GI50 MCF-7 = 27 nM; A431 = 45 nM
O
O
O
O
N
H
N
H
O
O
CO2H
O
R2
X
R1
N
H
X = NH; GI50 MCF-7 = 36 nM; A2780 = 62 nM;
A431 = 65 nM; Du145 = 51 nM; SMA = 17 nM
NH2
Norcantharidin acrylonitrile hybrids yields cytotoxic chimeric molecules with excellent activity against breast (MCF-7), skin (A431),
prostate (Du145), neuroblastoma (BE2-C) and pancreatic (MIA) carcinomas and against SMA (spontaneous murine astrocytoma).
10
This article is protected by copyright. All rights reserved.