Synthesis and anticancer activity of focused compound libraries from the natural product lead, oroidin

Article abstract of DOI:10.1016/j.bmc.2014.01.021

Oroidin (1), (E)-N-(3-(2-amino-1H-imidazol-4-yl)allyl)-4,5-dibromo-1H- pyrrole-2-carboxamide, is a pyrrole alkaloid isolated from the marine sponge Agelas oroides. Routine screening in a panel of twelve cancer cell lines revealed 1 to be poorly cytotoxic with the 50% growth inhibition concentration (GI₅₀) of 42 μM in MCF-7 (breast) cells and 24 μM in A2780 (ovarian) cells and >50 μM in all other cell lines tested. The development of eight focused libraries comprising thirty compounds total identified N-(biphenyl-4-ylmethyl)-1H-pyrrole-2-carboxamide (4l), N-benzyl-4,5-dibromo-1H- pyrrole-2-carboxamide (5a) and N-(biphenyl-4-ylmethyl)-4,5-dibromo-1H-pyrrole-2- carboxamide (5l) as potent inhibitors of cell growth in our panel of cell lines. Of these compounds GI₅₀ values of <5 μM were observed with 4l against HT29 (colon) and SW480 (colon); 5a against HT29; and 5l against HT29, SW480, MCF-7, A431 (skin), Du145 (prostate), BE2-C (neuroblastoma) and MIA (pancreas) cell lines. As a cancer class, colon cancer appears to be more sensitive to the oroidin series of compounds, with analogue 5l being the most active.

Full text of DOI:10.1016/j.bmc.2014.01.021

Bioorganic & Medicinal Chemistry 22 (2014) 1690–1699
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and anticancer activity of focused compound libraries from
the natural product lead, oroidin
Lauren Dyson a, Anthony D. Wright b, Kelly A. Young a, Jennette A. Sakoff c, Adam McCluskey a,
⇑
a Chemistry, Centre for Chemical Biology, School of Environmental & Life Sciences, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia
b Department of Pharmaceutical Sciences, College of Pharmacy, University of Hawaii at Hilo, 34 Rainbow Drive, Hilo, HI 96720, USA
c 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:
Oroidin (1), (E)-N-(3-(2-amino-1H-imidazol-4-yl)allyl)-4,5-dibromo-1H-pyrrole-2-carboxamide, is a pyr-
role alkaloid isolated from the marine sponge Agelas oroides. Routine screening in a panel of twelve cancer
Received 19 November 2013
Revised 7 January 2014
Accepted 15 January 2014
Available online 24 January 2014
cell lines revealed 1 to be poorly cytotoxic with the 50% growth inhibition concentration (GI50) of 42
lM
in MCF-7 (breast) cells and 24 M in A2780 (ovarian) cells and >50 M in all other cell lines tested. The
l
l
development of eight focused libraries comprising thirty compounds total identified N-(biphenyl-4-
ylmethyl)-1H-pyrrole-2-carboxamide (4l), N-benzyl-4,5-dibromo-1H-pyrrole-2-carboxamide (5a) and
N-(biphenyl-4-ylmethyl)-4,5-dibromo-1H-pyrrole-2-carboxamide (5l) as potent inhibitors of cell growth
Keywords:
Oroidin
Focused libraries
Cytotoxic compound
Natural product leads
Sponges
in our panel of cell lines. Of these compounds GI50 values of <5 lM were observed with 4l against HT29
(colon) and SW480 (colon); 5a against HT29; and 5l against HT29, SW480, MCF-7, A431 (skin), Du145
(prostate), BE2-C (neuroblastoma) and MIA (pancreas) cell lines. As a cancer class, colon cancer appears
to be more sensitive to the oroidin series of compounds, with analogue 5l being the most active.
Ó 2014 Elsevier Ltd. All rights reserved.
Synthesis
1. Introduction
Natural products and their derivatives account for almost half of
all approved drugs with the majority being of terrestrial origin.1,2
Many marine origin compounds are finding applications as
drugs,3,4 potential drugs,5 neutraceuticals,6 molecular tools,7
cosmeceuticals,8 agrochemicals,9 microbiological media (agar),10
and in the food and pharmaceutical industries.11 In terms of phar-
maceuticals some have shown potential as anticancer agents,5
antimicrobials,12 antimalarials13 and application in other medical
conditions.14–16 Our team has a long history of exploring a range
of natural product sources as a means to identify and develop novel
lead compounds against a range of human diseases.17,18 Indeed, we
have explored natural products targeting malaria, tuberculosis,
epilepsy and cancer. In this present study we re-isolated oroidin
(1), a natural product first isolated from the sponge Agelas oroides
in 1971 and later from several other sponges (Fig. 1).19,20
Figure 1. The chemical structure of oroidin (1), originally isolated from the marine
sponge Agelas oroides.21
Oroidin has also been used as a lead compound in the development
of inhibitors of bacterial biofilm formation.24 We have held a long
term interest in the discovery and development of cytotoxic small
molecules ranging from cantharidin analogues,25–28 cytotoxic ni-
triles through to mechanism based inhibition brought about by
dynamin and clathrin inhibitors.29–37 Given this interest, we
screened 1 against our panel of twelve cancer cell lines for growth
inhibition (see Table 1 for details). Oroidin displayed 42 and 24 lM
Oroidin (1) belongs to the ‘bromopyrrole’ family of alkaloids,
characterized by the presence of a brominated pyrrole–imidazole
moiety.22,23 While there are very few reports of the biological
activity associated with 1, it does display modest levels of anti-pro-
tozoal activity against Trypanosoma brucei rhodesiense, Trypano-
soma cruzi, Leishmania donovani and Plasmodium falciparum.23
potency against MCF-7 (breast) and A2780 (ovarian) carcinoma
cell lines, respectively. While only moderately potent at inhibiting
cell growth, oroidin’s structural simplicity suggested that rapid
elaboration might be possible.
While structurally simple, the reported synthetic routes to
oroidin include Pd-mediated coupling with preformed imidazole
moieties,38–42 approaches requiring amino acid precursors43–47
and olefination strategies.48–52 More recently Rasapalli et al.,
reported the use of imidazo[1,2-a]pyrimidines as a heterocyclic
⇑
Corresponding author. Tel.: +61 249 216486; fax: +61 249 215472.
E-mail address: Adam.McCluskey@newcastle.edu.au (A. McCluskey).
http://dx.doi.org/10.1016/j.bmc.2014.01.021
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

L. Dyson et al. / Bioorg. Med. Chem. 22 (2014) 1690–1699
1691
Table 1
Percentage (%) growth inhibition in response to 25
lM of compounds in oroidin series from Library 1 and 2 (4a–d and 5a–d)
Compound
HT29a
SW480a
MCF-7b
A2780c
H460d
A431e
Du145f
BE2-Cg
SJ-G2h
MIAi
SMAh
U87h
4a-d
5a-d
1
31
9
3
8
1
27
3
6
5
51
6
2
7
4
6
2
7
23
5
6
5
<0
<0
21
2
15
5
3
3
Oroidin
4a
5a
7
6
33
4
4
<0
84
3
43
<0
56
5
52
9
3
<0
<0
33
<2
61
3
31
5
8
>100
>100
>100
95
2
4
6
2
4b
5b
9
46
5
2
9
1
7
5
7
70
7
1
6
58
4
3
5
7
5
7
2
0.4
5
<0
11
<0
62
2
22
9
3
<0
19
7
53
3
7
9
<0
4
1
19 10
6
4c
5c
16
41
5
1
7
<0
5
21
63
2
5
10
48
5
4
13
25
6
8
1
5
4
2
<0
<0
<0
<0
0
8
4
3
10 0.2
21
8
45
2
9
11
<0
2
4d
5d
16
41
2
2
<0
<0
23
70
4
1
13
56
3
5
11
17
3
6
12
24
2
3
10
19
6
5
<0
<0
8
14
3
8
15
34
5
4
6
40
1
5
<0
<0
a
b
c
d
e
f
Colon.
Breast.
Ovarian.
Lung.
Skin.
Prostate.
Neuroblastoma.
Glioblastoma.
Pancreas.
g
h
i
surrogate for the polyfunctionalised 2-amino-1H-imidazole nu-
cleus of oroidin, hymenidin and clathordin.53 Takale et al. have also
recently reported the synthesis and anti-bacterial activity of oroi-
din analogues.54 The biosynthesis and synthesis of this natural
product containing the pyrrolic ring has been reviewed by Al-
Mourabit55 and Young.56 To date all methods of oroidin synthesis
found within the literature have been either complex, not stereo-
specific and typically low yielding.
In our initial design approaches we viewed the C@C as only
serving to increase synthetic complexity. Thus in the synthesis of
our focused libraries, only linker saturated analogues were consid-
ered. In this initial report we restricted the choice of pyrrole moiety
to the 4,5-dibromo and the parent pyrrole, a Br to H isosteric
modification. We envisaged a simple nucleophilic displacement
approach to the coupling of a family of amines with either the
2,2,2-trichloro-1-(1H-pyrrol-2-yl)ethanone (2) or 2,2,2-trichloro-
1-(4,5-dibromo-1H-pyrrol-2-yl)ethanone (3) which are commer-
cially available.44,45 Judicious choice of amine would allow
investigation of Region B and C: the linker and tail group. In the
assembly of the focused libraries associated with the chosen Re-
gion A moieties, the amines were characterized as: unsubstituted
phenyl analogues (Library 1 and 2), substituted phenyl analogues
(Library 3 and 4), sterically bulky analogues (Library 5 and 6),
and imidazole analogues (Library 7 and 8) where Libraries 1, 3, 5,
7 and 2, 4, 6, 8 correspond to the pyrrole and 4,5-dibromopyrrole
head groups respectively (Fig. 3).
2. Results
As the initial activity of 1 was modest we examined structural
modifications; however, current literature approaches were
viewed as too complex for focused library development, validation
and potential enhancement of the cytotoxicity of oroidin-based
analogues, even though the recent synthesis by Rasapalli now al-
lowed for more rapid access into the oroidin scaffold.53 Adopting
a simpler approach we viewed 1 as comprising three modifiable re-
gions (A) the pyrrole moiety tail group, (B) a central linker, and (C)
the aminoimidazole head group connected via a central linker con-
taining an olefinic moiety (Fig. 2).
Our initial synthetic efforts investigated the displacement of the
trichloroethanone moiety by treatment of 2 with benzylamine in
DMF with two equivalents of Et3N at room temperature. This
Figure 2. The three modifiable regions and retrosynthetic analysis of oroidin: the oroidin molecule, divided into three sections: Region A—the 4,5-dibromo-1H-pyrrole tail
group; Region B—the alkenyl linker; and Region C—the head group. Oroidin analogues can be accessed via simple coupling of an amine with the pyrrole trichloromethyl
ketone.

1692
L. Dyson et al. / Bioorg. Med. Chem. 22 (2014) 1690–1699
Figure 3. Amines used to develop focused Libraries 1–8. Libraries 1, 3, 5, and 7 were synthesized using 4,5-dibromopyrrole 2-carboxaldehyde and amines a–o; and Libraries
2, 4, 6 and 8 were synthesized using pyrrole 2-carboxaldehyde and amines a–o.
resulted in a low (30%) conversion after 24 h. Increasing the
reaction temperature to 60 °C gave 70% conversion (by 1H NMR).
However, the isolated yield of this product was typically <50% after
removal of DMF and subsequent column chromatography. Further
increases in temperature as well as the use of microwave heating
did not afford any further yield enhancements, and resulted in
product decomposition. Changing the solvent to acetonitrile and
increasing the amount of Et3N to five equivalents afforded a sim-
pler workup with an isolated yield of 65% at 60 °C, with overnight
stirring (see Section 4) (see Scheme 1).
Of the oroidin library analogues synthesized only the histamine
analogues (4o and 5o) were unable to be isolated using the general
method described above. In both instances the starting amine was
used as the hydrochloride salt. The use of piperidine (rather than
Et3N) allowed these materials to be produced as required without
first isolating the free amine. The isolated yields of the Library ana-
logues ranged from poor (13%; 5n) to excellent (98%, 4l).
highest level of growth inhibition noted with 4c/d towards the
MCF-7 cell line, and this was a very modest 21 and 23%, respec-
tively. Library 2, the dibromo substituted library showed more
promise with 5a displaying growth inhibition levels appropriate
for full GI50 determination. Complete growth inhibition was noted
with HT29 (colon), MCF-7 (breast) and BE2-C (neuroblastoma) cell
lines. The introduction of methylene spacers through the synthesis
of 5b–d served only to reduce the observed cytotoxicity in a linker
length dependent manner.
We identified seven analogues 5a (Library 2; Table 1), 5e, 5f,
and 5i (Library 4, Table 2), 4l (Library 5; Table 3), 5k and 5l (Library
6; Table 3) with a level of growth inhibition sufficient to warrant
progression to GI50 determination. Surprisingly, no analogues from
Library 7 or 8 displayed any noteworthy activity or retained the
modest activity against MCF-7 and A2780 cell lines observed with
1. With the exception of 4l, all active compounds contained the
4,5-dibromopyrrole moiety suggesting a key role for the bromine
moieties in eliciting the observed cytotoxic response.
All oroidin analogues were tested for their ability to inhibit
cancer cell growth in a panel of 12 cancer cell lines: colon
(HT29, SW480), skin (A431), lung (H460), ovarian (A2780), breast
(MCF-7), prostate (Du145), pancreatic (MIA), glioblastoma (SJ-G2,
SMA, U87) and neuroblastoma (BE2-C). Initial screening was con-
Within the substituted phenyl group the mono (5e) and disub-
stituted (5f) chlorine analogues maintained roughly the same
modest activity with little selectivity across the cell lines tested
returning average GI50 values of 23.9 and 19.9
Lengthening the chain and replacing the chlorine with a hydroxyl
group (5h) had little effect (average GI50 = 31 M); however, po-
lM, respectively.
ducted in triplicate at a single drug concentration of 25 lM. The
outcome of this screening is shown in Tables 1–4. Compounds
that demonstrated 90% inhibition in at least one of the cell lines
analyzed, or a consistently high level of cell death across the cell
lines evaluated, were subjected to full dose response evaluation
(growth inhibition, GI50). The outcome of this screening is shown
in Table 5.
l
tency was lost in a few of the cell lines (SW480, DU145 and
U87). Most interesting were the analogues lacking the para substi-
tuted functionality (5a) that displayed a good to average level of
cytotoxicity (average GI50 = 24.8 lM) suggesting that an aromatic
substituent was not a pre-requisite for activity in this screen. This
compound also lost potency in the same cell lines but gained po-
tency towards the HT29 and BE2-C cell lines with GI50 values
Analysis of the initial 25 lM drug screen with Library 1 shows
essentially no activity in any of the cell lines examined with the
<5
l
M.
The sterically bulky diphenyl analogue (4l) followed the same
trend as the chlorinated analogues discussed above with modest
activity across all cell lines tested, with an average GI50 value of
15.6
fied in this study. The biphenyl analogue (5l) however, displayed
the most potency, with an average GI50 value of 13.9 M, of all
lM making it the second most active oroidin analogue identi-
l
compounds tested suggesting the linear, twisted conformation of
the biphenyl head group is more favorable in comparison to the
‘paddle-like’ conformation of the diphenyl head group.
Scheme 1. Reagents and conditions. (i) CH3CN, Et3N (5 equiv), 65 °C 24 h.

L. Dyson et al. / Bioorg. Med. Chem. 22 (2014) 1690–1699
1693
Table 2
Percentage growth inhibition in response to 25
l
M of compounds in oroidin series from Library 3 and 4 (4e–i and 5e–i)
Compound
HT29a
SW480a
MCF-7b
A2780c
H460d
A431e
Du145f
BE2-Cg
SJ-G2h
MIAi
SMAh
U87h
4e-i
5e-i
1
31
3
8
1
27
6
51
6
7
4
2
6
2
1
23
<0
5
6
5
<0
<0
21
2
15
5
3
3
Oroidin
4e
5e
10
95
3
2
4
11
2
3
10
>100
2
6
2
86
7
1
<0
57
<0
37
<0
24 10
1
63
2
0
10
79
3
2
4
30
2
2
40 10
42
5
4f
5f
14
77
4
1
<0
22
7
>100
15
90
5
1
14
66
3
6
6
57
5
7
<0
66
<0
44
11 10
70
<0
77
17
84
6
1
4
55
6
3
4
5
6
2
1
3
4g
5g
3
14
5
4
10 11
<0
5
30
4
5
6
48
0.4
2
2
13
4
2
<0
12
8
<0
<0
<0
<0
4
6
14
3
3
<0
23
<0
<0
3
4
3
1
4
4h
5h
3
48
6
2
5
<0
5
4
8
81
7
3
18
90
7
2
2
11
1
4
<0
68
<0
3
<0
11 13
44
6
20
6
3
<1
46
2
<0
3
9
101
2
8
4i
5i
22
81
3
1
<0
5
19
88
4
2
<0
67
3
23
3
2
0
22
5
8
1
7
8
4
<0
<0
22
12
31
4
1
8
79
3
1
0
6
4
4
1
20 10
3
a
b
c
d
e
f
Colon carcinoma.
Breast carcinoma.
Ovarian.
Lung.
Skin.
Prostate.
g
h
i
Neuroblastoma.
Glioblastoma.
Pancreas.
Remarkably, the change from a phenyl (5a) to a biphenyl (5l)
caused significant changes in selectivity. Analogue 5l retained high
activity against the colon (HT29; 5l GI50 = 3.4 1.3 vs 5a
noteworthy levels of cell death identified a number of promising
cytotoxic compounds. Of the thirty analogues seven (4l, 5a, 5e,
5f, 5h, 5k and 5l) proceeded to full dose response evaluation. The
lead compound, oroidin (1), was also evaluated and returned poor
GI50 = 4.6 0.1
GI50 = 4.8 0.3
l
l
M) and neuronal (BE2-C; GI50 = 4.2 1.2 vs 5a
M) cell lines but activity increased at least ten fold
levels of cytotoxicity with GI50 values >50
excepting MCF-7 (breast; 42 M) and A2780 (ovarian;
24 M). Out of the seven novel analogues subjected to full dose
response the average GI50 values were 15.6, 24.8, 23.9, 19.9, 31,
19.1 and 13.9 M for 4l, 5a, 5e, 5f, 5h, 5k and 5l, respectively. Of
these GI50 values of <5 M were observed with 4l against HT29
lM for all cell lines
towards the SW480 (5l GI50 = 2.4 0.4 vs 5a GI50 >50
(5l GI50 = 2.6 0.7 vs 5a GI50 >50 M) and MIA (5l GI50 = 3.4 0.8
vs 5a GI50 >50 M) cell lines, threefold towards two others, MCF-
(5l GI50 = 3.1 1.1 vs 5a GI50 = 11 M) and A431 (5l
GI50 = 3.7 0.5 vs 5a GI50 = 22 0.3 M), but decreased five-fold to-
wards SJ-G2 (5l GI50 >50 vs 5a GI50 = 11 0.0 M) cells. Both 5l and
lM), Du145
4 l
l
4 l
l
7
2
l
l
l
l
l
and SW480; 5a against HT29; and 5l against HT29, SW480, MCF-
7, A431, Du145, BE2-C and MIA cell lines. As a cancer class, colon
cancer appears to be more sensitive to the oroidin class of com-
pounds, with analogue 5l being the most active oroidin analogue
developed in this work.
4l showed selectivity against the colon (SW480) cell line in compar-
ison to the other compounds tested. The two interesting results are
observed in the lung (H460; 4l GI50 >50 vs 5l GI50 = 21 10
lM))
and brain (U87; 4l GI50 = 11 1 vs 5l average GI50 >50 M) cell
l
lines. Here removal of the bromine atoms leads to a more than
50% reduction in activity towards lung (H460) but a more than a
five-fold increase in activity towards the brain (U87) cell line.
The combination of structural simplicity, ease of synthetic
manipulation and promising cytotoxicity of 5l in particular means
that oroidin analogues are promising leads for the development of
potentially novel anticancer drugs. Currently the mechanism of
cytotoxic action is unknown. Thus our current efforts are targeted
towards elucidations of the mechanism of action, through chemical
biology approaches to potentially identify the in cell target of oroi-
din analogues and the possible enhancement of potency through
rational drug design approaches. We will report our findings in this
area in due course.
3. Conclusions
Herein we have synthesized thirty analogues of the natural
product oroidin (1). Screening of these compounds against a panel
of twelve cancer cell lines, initially at a drug concentration of
25 lM, with subsequent dose response evaluation for those with

1694
L. Dyson et al. / Bioorg. Med. Chem. 22 (2014) 1690–1699
Table 3
Percentage growth inhibition in response to 25
l
M of compounds in oroidin series from Library 5 and 6 (4j–l and 5j–l)
Compound
HT29a
SW480a
MCF-7b
A2780c
H460d
A431e
Du145f
BE2-Cg
SJ-G2h
MIAi
SMAh
U87h
4j-l
5j-l
1—Oroidin
31
3
8
1
1
27
6
51
6
7
4
6
2
23
5
6
5
<0
21
2
15
5
3
3
4j
5j
19
28
2
5
3
<0
23
53
3
5
13
52
8
3
23
12
3
2
8
15
1
4
20
14
2
6
<0
<0
8
10
3
9
20 13
21
16
17
4
1
<1
<0
2
4k
5k
29
82
3
1
<0
33
52
>100
5
27
94
3
1
11
67
1
1
30
56
9
8
8
56
10
2
<0
>100
53
56
7
8
32
69
1
3
15
80
4
4
14
48 10
4
6
4l
5l
78
78
2
2
93
97
2
1
79
70
1
2
58
66
2
1
59
61 0.1
3
81
90
2
2
87
64
4
9
72
57
3
3
33
17
8
4
74 0.5
82
53
59
5
3
77
58
3
6
2
a
b
c
d
e
f
Colon carcinoma.
Breast carcinoma.
Ovarian.
Lung.
Skin.
Prostate.
g
h
i
Neuroblastoma.
Glioblastoma.
Pancreas.
Table 4
Percentage growth inhibition in response to 25
l
M of compounds in oroidin series from Library 7 and 8 (4m–o and 5m–o)
Compound
HT29a
SW480a
MCF-7b A2780c H460d A431e Du145f BE2-Cg SJ-G2h
MIAi
SMAh
U87h
4m-o
5m-o
1—Oroidin
31
3
8
1
1
27
6
4
51
6
7
4
6
2
23
5
6
5
<0
21
19
2
1
15
5
3
3
2
4m
5m
4
4
4
3
9
<0
7
12
4
10
15
6
5
14
14
2
5
1
3
3
6
7
5
2
11
1
6
3
3
<0
<0
8
6
5
0
9
<0
3
0
4n
5n
5
10
2
7
7
26
5
5
8
36
6
1
21
7
3
12
<0
11
5
28
7
3
<0
20
<0
10
4
15
8
<0
11
3
<0
2
9
6
3
5
22 11
8
4
4
5
6
4
7
4o
5o
11
11
4
5
1
12
1
23
3
11
37
3
3
0
35
1
3
2
4
2
5
<0
5
<0
13 10
<0
11
7
20
7
0
18
2
6
5
6
2
3
2
2
a
b
c
d
e
f
Colon carcinoma.
Breast carcinoma.
Ovarian.
Lung.
Skin.
Prostate.
g
h
i
Neuroblastoma.
Glioblastoma.
Pancreas.

L. Dyson et al. / Bioorg. Med. Chem. 22 (2014) 1690–1699
1695
Table 5
The GI50 values determined for the most active compounds in the oroidin series
Compound
HT29a
SW480a
MCF-7b
A2780c
H460d
A431e
GI50
Du145f
lM)
BE2-Cg
>50
SJ-G2h
>50
MIAi
>50
SMAh
>50
U87h
(
1
>50
>50
42
4
24
11
4
3
>50
>50
>50
>50
>50
Oroidin
4l
5a
5e
5f
5h
5k
5l
4.3 0.9
4.6 0.1
4.3 0.5
>50
5.7
11
2
2
5.8 0.2
22 0.3
6.5
33
27
1
3
3
11
2
>50
11 0.0
6.5 0.7
37 1.2
22
17 0.6
43
21
25
16
5
2
2
11
1
14 2.1
35
28 0.3
19
3
4.8 0.3
32 0.3
>50
34
27
13
1
36
33
2
3
14 0.9
13
15
11
13
2
2
1
1
24
20
3
1
28
2
3
2
1
17 0.7
28 0.3
16 0.6
3.4 1.3
12
12
0
0
1
20 0.7
>50
24
16 0.3
16
4.2 1.2
3
18 0.9
30 0.6
17 0.3
23 0.7
11 0.3
>50
34
39 0.6
18 0.3
21 10
20 0.6
19 0.2
3.7 0.5
1
>50
36
2
10 0.7
3.1 1.1
21
2
2
27
4
18 0.7
3.4 0.8
4
2.4 0.4
6.4 1.8
2.6 0.7
>50
17
3
>50
a
b
c
d
e
f
Colon carcinoma.
Breast carcinoma.
Ovarian.
Lung.
Skin.
Prostate.
g
h
i
Neuroblastoma.
Glioblastoma.
Pancreas.
supplemented with 10% fetal bovine serum, 10 mM sodium bicar-
4. Experimental
4.1. General techniques and instrumentation
bonate penicillin (100 IU/mL), streptomycin (100
tamine (4 mM).
lg/mL), and glu-
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
All reagents were purchased from Sigma Aldrich, Matrix Scien-
tific, AK Scientific, and Merck Scientific, and used without further
purification unless otherwise stated.
1H and 13C NMR spectra were recorded on a Bruker Avance™
AMX 400 MHz spectrometer at 400.13 and 100.62 MHz, respec-
tively. Spectra were recorded using deuterated acetone (Acetone-
d6) and deuterated dimethylsulfoxide (DMSO-d6), as specified.
For acetone-d6 the residual solvent peaks were used as the internal
reference [d 2.05 (quintet) and 29.8 ppm (septet) for 1H and 13C
NMR, respectively]. For DMSO-d6 the residual solvent peaks were
used as the internal reference [d 2.50 (quintet) and 39.5 ppm (sep-
tet) for 1H and 13C NMR, respectively]. Chemical shifts (d) are re-
ported in parts per million (ppm) measured relative to the
internal references, and coupling constants (J) are expressed in
Hertz (Hz). Multiplicities are denoted as singlet (s), broad singlet
(br s) doublet (d), doublet of doublets (dd), triplet (t), doublet of
triplets (dt), quartet (q), quintet (quintet) and multiplet (m). Peaks
are listed according to the following convention: chemical shift,
multiplicity, coupling constant and integration.
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
well. After 72 h drug exposure growth inhibitory effects were eval-
uated using the MTT (3-[4,5-dimethyltiazol-2-yl]-2,5-diphenyl-
tetrazolium bromide) assay and absorbance read at 540 nm. Per-
centage growth inhibition was determined at a fixed drug concen-
lL medium at a density of 2500–4000 cells/well. On day 0,
l
tration of 25 lM. A value of 100% is indicative of total cell growth
inhibition. Those analogues showing appreciable percentage
growth inhibition underwent further dose response analysis allow-
ing for the calculation of a GI50 value. This value is the drug concen-
tration at which cell growth is inhibited by 50% based on the
difference between the optical density values on day 0 and those
at the end of drug exposure.57
4.2. Synthesis of oroidin analogues
Melting points were recorded on a Buchi Melting Point M-565
system. Thin layer chromatography (TLC) was performed on Merck
silica gel 60 F254 pre-coated aluminum plates with a thickness of
0.2 mm. Visualization was achieved using UV light followed by
4.2.1. General procedure 1
4.2.1.1. Formation of N-Benzyl-1H-pyrrole-2-carboxamide (4a)
2,2,2-Trichloro-1-(1H-pyrrol-2-yl)ethanone (2) (0.200 g, 0.94 mmol)
and benzylamine (0.101 g, 0.11 mL, 0.94 mmol) were dissolved in
acetonitrile (15.0 mL). Triethylamine (5 equiv, 0.65 mL) was added
to the solution, and the reaction mixture stirred at 60 °C for 24 h.
Solvent was removed from the reaction by rotary evaporation.
The resulting yellow oil was purified using 5% MeOH/CH2Cl2 to
afford N-benzyl-1H-pyrrole-2-carboxamide (0.123 g, 65%) as a
white solid, mp 128–130 °C.
dipping plates in
a solution of phosphomolybdic acid (2.5%
H2SO4, 2% H3PO3MoO4, 1% Ce(SO4)2) and heating.
Microwave reactions were conducted in a 10 mL glass micro-
wave vessel using a CEM Discover microwave reactor.
Column chromatography was performed under ‘flash’ condi-
tions on Merck silica gel 60 (230–400 mesh).
Low resolution mass spectrometry (LRMS) was conducted on a
Shimadzu LCMS-2010EV quadrupole mass spectrometer. Accurate
masses (HRMS) were determined using an Applied Biosystems Q-
STAR XL mass spectrometer.
1H NMR (DMSO-d6): d 11.45 (s, 1H), 8.52 (t, J = 5.9 Hz, 1H),
7.36–7.27 (m, 4H), 7.26–7.20 (m, 1H), 6.85 (br s, 1H), 6.82 (br s,
1H), 6.12–6.03 (m, 1H), 4.43 (d, J = 6.1 Hz, 2H).
13C NMR (DMSO-d6): d 160.6, 140.1, 128.2 (2 Â C), 127.1 (2 Â C),
126.7, 126.1, 121.4, 110.0, 108.6, 41.8.
4.1.1. Cell culture and stock solutions
Stock solutions were prepared as follows and stored at À20 °C:
drugs were stored as 30 mM solutions in DMSO. All cell lines were
cultured at 37 °C, under 5% CO2 in air and were maintained in
Dulbecco’s modified Eagle’s medium (Trace Biosciences, Australia)
4.2.1.2. N-Phenethyl-1H-pyrrole-2-carboxamide (4b). Com-
pound 4b was synthesized according to general procedure 1 from
2
(0.200 g, 0.94 mmol) and 2-phenylethanamine (0.114 g,

1696
L. Dyson et al. / Bioorg. Med. Chem. 22 (2014) 1690–1699
0.12 mL, 0.94 mmol). The resulting clear oil was purified using col-
umn chromatography from 2% MeOH/CH2Cl2 to afford 4b (0.037 g,
19%) as a yellow solid, mp 132–134 °C.
column chromatography from 50% EtOAc/Hexane to afford 4g
(0.044 g, 20%) as a white solid, mp 110–113 °C.
1H NMR (DMSO-d6): d 11.42 (s, 1H), 8.44 (t, J = 5.9 Hz, 1H), 7.22
(d, J = 8.5 Hz, 2H), 6.92–6.82 (m, 3H), 6.80 (s, 1H), 6.07 (dd, J = 5.5,
2.3 Hz, 1H), 4.35 (d, J = 6.0 Hz, 2H), 3.72 (s, 3H).
1H NMR (DMSO-d6): d 11.38 (s, 1H), 8.05 (t, J = 5.5 Hz, 1H),
7.34–7.26 (m, 2H), 7.26–7.15 (m, 3H), 6.83 (br s, 1H), 6.72 (br s,
1H), 6.09–6.02 (m, 1H), 3.47–3.38 (m, 2H), 2.81 (t, J = 7.5 Hz, 2H).
13C NMR (DMSO-d6): d 160.6, 139.6, 128.6 (2 Â C), 128.3 (2 Â C),
126.4, 126.0, 121.1, 109.7, 108.4, 35.5, 30.7.
13C NMR (DMSO-d6): d 160.5, 158.1, 132.0, 128.5 (2 Â C), 126.2,
121.3, 113.6 (2 Â C), 109.9, 108.5, 55.0, 41.2.
HRMS (ESI) m/z: calculated for C13H15N2O2 [M+H]+ 231.1128;
Found 231.1131.
4.2.1.3. N-(3-Phenylpropyl)-1H-pyrrole-2-carboxamide (4c)
Compound 4c was synthesized according to general procedure 1
4.2.1.8. N-(4-Hydroxyphenethyl)-1H-pyrrole-2-carboxamide (4h)
Compound 4h was synthesized according to general procedure 1
from
2
(0.200 g, 0.94 mmol) and 3-phenylpropan-1-amine
(0.127 g, 0.13 mL, 0.94 mmol). The resulting clear oil was purified
using column chromatography from 1% MeOH/CH2Cl2 to afford
4c (0.164 g, 76%) as a white solid, mp 88–90 °C.
from
2
(0.200 g, 0.94 mmol) and 4-(2-aminoethyl)phenol
(0.116 g, 0.94 mmol). The resulting orange oil was purified using
column chromatography from 2% MeOH/CH2Cl2 to afford 4h
(0.104 g, 48%) as a white solid, mp 143–146 °C.
1H NMR (DMSO-d6): d 11.39 (s, 1H), 7.98 (t, J = 5.5 Hz, 1H),
7.30–7.15 (m, 5H), 6.86–6.80 (m, 1H), 6.79–6.71 (m, 1H), 6.10–
3.04 (m, 1H), 3.23 (q, J = 6.6 Hz, 2H), 2.61 (t, J = 7.7 Hz, 2H), 1.79
(p, J = 7.4 Hz, 2H).
1H NMR (DMSO-d6): d 11.37 (s, 1H), 9.16 (s, 1H), 8.01 (t,
J = 5.4 Hz, 1H), 7.01 (d, J = 8.4 Hz, 2H), 6.86––6.79 (m, 1H), 6.72
(br s, 1H), 6.68 (d, J = 8.4 Hz, 2H), 6.11–6.02 (m, 1H), 3.40–3.31
(m, 2H, obscured by water signal), 2.68 (t, J = 7.5 Hz, 2H).
13C NMR (DMSO-d6): d 160.6, 155.6, 129.7, 129.5 (2 Â C), 126.4,
121.1, 115.1 (2 Â C), 109.7, 108.5, 40.5, 34.7.
13C NMR (DMSO-d6): d 160.6, 141.8, 128.3 (2 Â C), 128.3 (2 Â C),
126.4, 125.7, 121.1, 109.6, 108.4, 38.1, 32.6, 31.2.
4.2.1.4.
N-(4-Phenylbutyl)-1H-pyrrole-2-carboxamide
(4d)
HRMS (ESI) m/z: calculated for C13H15N2O2 [M+H]+ 231.1128;
Found 231.1133.
Compound 4d was synthesized according to general procedure 1
from 2 (0.200 g, 0.94 mmol) and 4-phenylbutylamine (0.140 g,
0.15 mL, 0.94 mmol) were dissolved in acetonitrile (15.0 mL). The
resulting clear yellow oil was purified using column chromatogra-
phy from 50% EtOAc/Hexane to afford 4d (0.151 g, 66%) as a white
solid, mp 109–112 °C.
4.2.1.9. N-(4-Ethylphenethyl)-1H-pyrrole-2-carboxamide (4i)
Compound 4i was synthesized according to general procedure 1
from 2 (0.200 g, 0.94 mmol) and (4-ethylphenyl)methanamine
(0.127 g, 0.15 mL, 0.94 mmol). The resulting white solid was puri-
fied from column chromatography from 1% MeOH/CH2Cl2 to afford
4i (0.081 g, 36%) as an off-white solid, mp 166–167 °C.
1H NMR (DMSO-d6): d 11.38 (s, 1H), 8.04 (t, J = 5.5 Hz, 1H),
7.16–7.11 (m, 4H), 6.83 (br s, 1H), 6.72 (br s, 1H), 6.09–6.03 (m,
1H), 3.40 (q, J = 6.9 Hz, 2H), 2.77 (t, J = 7.4 Hz, 2H), 2.56 (q,
J = 7.6 Hz, 2H), 1.16 (t, J = 7.6 Hz, 3H).
1H NMR (DMSO-d6): d 11.37 (s, 1H), 7.94 (t, J = 5.5 Hz, 1H),
7.30–7.22 (m, 2H), 7.22–7.11 (m, 3H), 6.82 (br s, 1H), 6.74 (br s,
1H), 6.11–6.01 (m, 1H), 3.23 (q, J = 6.5 Hz, 2H), 2.60 (t, J = 7.4 Hz,
2H), 1.65–1.55 (m, 2H), 1.55–1.45 (m, 2H).
13C NMR (DMSO-d6): d 160.6, 142.2, 128.3 (2 Â C), 128.2 (2 Â C),
126.5, 125.6, 121.0, 109.6, 108.4, 38.2, 34.9, 29.1, 28.5.
13C NMR (DMSO-d6): d 160.6, 141.4, 136.8, 128.5 (2 Â C), 127.7
(2 Â C), 126.4, 121.1, 109.7, 108.4, 35.1, 30.7, 27.8, 15.7.
HRMS (ESI) m/z: calculated for C15H19N2O [M+H]+ 243.1492;
Found 243.1499.
4.2.1.5. N-(4-Chlorobenzyl)-1H-pyrrole-2-carboxamide (4e)
Compound 4e was synthesized according to general procedure 1
from 2 (0.200 g, 0.94 mmol) and (4-chlorophenyl)methanamine
(0.133 g, 0.12 mL, 0.94 mmol). The resulting clear oil was purified
using column chromatography from 2% MeOH/CH2Cl2 to afford
4e (0.063 g, 29%) as a white solid, mp 126–128 °C.
4.2.1.10. N-(2-(Naphthalen-2-yl)ethyl)-1H-pyrrole-2-carboxam-
ide (4j).
Compound 4j was synthesized according to general
1H NMR (Acetone-d6): d 10.97 (s, 1H), 8.02 (br s, 1H), 7.39–7.29
(m, 4H), 6.99–6.91 (m, 1H), 6.86–6.79 (m, 1H), 6.18–6.11 (m, 1H),
4.54 (d, J = 6.2 Hz, 2H).
procedure 1 from 2 (0.200 g, 0.94 mmol) and 2-(naphthalen-2-
yl)ethanamine (0.161 g, 0.94 mmol). The resulting orange oil was
purified using column chromatography from 1% MeOH/CH2Cl2 to
afford 4j (0.126 g, 51%) as a white solid, mp 141–144 °C.
1H NMR (DMSO-d6): d 11.39 (s, 1H), 8.10 (t, J = 5.6 Hz, 1H),
7.90–7.82 (m, 3H), 7.74 (s, 1H), 7.50–7.39 (m, 3H), 6.86–6.80 (m,
1H), 6.73 (br s, 1H), 6.09–6.03 (m, 1H), 3.54 (q, J = 6.8 Hz, 2H),
2.99 (t, J = 7.3 Hz, 2H).
13C NMR (Acetone-d6): d 162.0, 139.9, 132.9, 130.1 (2 Â C),
129.1 (2 Â C), 127.1, 122.4, 110.4, 109.8, 42.6.
4.2.1.6. N-(3,4-Dichlorobenzyl)-1H-pyrrole-2-carboxamide (4f)
Compound 4f was synthesized according to general procedure 1
13C NMR (DMSO-d6): d 160.6, 137.3, 133.2, 131.7, 127.7, 127.5,
127.5, 127.3, 126.6, 126.4, 126.0, 125.3, 121.1, 109.7,108.5, 40.0, 35.7.
HRMS (ESI) m/z: calculated for C17H17N2O [M+H]+ 265.1335;
Found 265.1327.
from
2
(0.200 g, 0.94 mmol) and 3,4-dichlorobenzylamine
(0.165 g, 0.94 mmol). The resulting pink oil was purified using col-
umn chromatography from 1% MeOH/CH2Cl2 to afford 4f (0.106 g,
42%), mp 124–126 °C.
1H NMR (DMSO-d6): d 11.48 (s, 1H), 8.59 (t, J = 6.0 Hz, 1H), 7.58
(d, J = 8.3 Hz, 1H), 7.53 (d, J = 1.3 Hz, 1H), 7.29 (dd, J = 8.3, 1.4 Hz,
1H), 6.87 (br s, 1H), 6.81 (br s, 1H), 6.13–6.07 (m, 1H), 4.41 (d,
J = 6.1 Hz, 2H).
4.2.1.11. N-(2,2-Diphenylethyl)-1H-pyrrole-2-carboxamide (4k)
Compound 4k was synthesized according to general procedure 1
from
2
(0.200 g, 0.94 mmol) and 2,2-diphenylethanamine
13C NMR (DMSO-d6): d 160.8, 141.4, 130.8, 130.5, 129.1, 129.1,
127.5, 125.8, 121.7, 110.2, 108.7, 40.9.
(0.185 g, 0.94 mmol). The resulting orange oil was purified using
column chromatography from 1% MeOH/CH2Cl2 to afford 4k
(0.183 g, 67%) as a white solid, mp 180–182 °C.
1H NMR (DMSO-d6): d 11.36 (s, 1H), 8.00 (t, J = 5.6 Hz, 1H),
7.33–7.26 (m, 8H), 7.20–7.14 (m, 2H), 6.86–6.76 (m, 1H), 6.66–
6.59 (m, 1H), 6.06–5.98 (m, 1H), 4.37 (t, J = 7.8 Hz, 1H), 3.85 (dd,
J = 7.8, 5.8 Hz, 2H).
4.2.1.7. N-(4-Methoxybenzyl)-1H-pyrrole-2-carboxamide (4g)
Compound 4g was synthesized according to general procedure 1
from 2 (0.200 g, 0.94 mmol) and 4-methoxybenzylamine (0.129 g,
0.12 mL, 0.94 mmol). The resulting pink oil was purified using

L. Dyson et al. / Bioorg. Med. Chem. 22 (2014) 1690–1699
1697
13C NMR (DMSO-d6): d 160.6, 143.0, 128.4 (4 Â C), 128.0 (4 Â C),
126.3 (2 Â C), 126.2 (2 Â C), 121.2, 109.9, 108.4, 50.3, 43.1.
HRMS (ESI) m/z: calculated for C19H19N2O [M+H]+ 291.1492;
Found 291.1496.
13C NMR (DMSO-d6): d 160.6, 134.7, 126.4, 121.2 (2 Â C), 109.7,
108.5, 38.7, 27.4.
HRMS (ESI) m/z: calculated for C10H13N4O [M+H]+ 205.1084;
Found 205.1095.
4.2.1.12.
N-(Biphenyl-4-ylmethyl)-1H-pyrrole-2-carboxamide
4.2.2. General procedure 2
(4l). Compound 4l was synthesized according to general proce-
dure 1 from 2 (0.200 g, 0.94 mmol) and biphenyl-4-ylmethan-
amine (0.100 g, 0.54 mmol). The resulting yellow solid was
purified using column chromatography from 1% MeOH/CH2Cl2 to
afford 4l (0.147 g, 98%) as a white solid, mp 125–128 °C.
1H NMR (DMSO-d6): d 11.46 (s, 1H), 8.55 (t, J = 6.0 Hz, 1H),
7.65–7.59 (m, 4H), 7.47–7.41 (m, 2H), 7.40–7.30 (m, 3H), 6.86 (br
s, 1H), 6.83 (br s, 1H), 6.12–6.06 (m, 1H), 4.46 (d, J = 6.0 Hz, 2H).
13C NMR (DMSO-d6): d 160.7, 140.0, 139.3, 138.6, 128.9 (2 Â C),
127.8 (2 Â C), 127.3, 126.6 (2 Â C), 126.6 (2 Â C), 126.2, 121.4,
110.0, 108.6, 41.6.
4.2.2.1. N-Benzyl-4,5-dibromo-1H-pyrrole-2-carboxamide (5a)
2,2,2-Trichloro-1-(4,5-dibromo-1H-pyrrol-2-yl)ethanone
(3)
(0.200 g, 0.54 mmol), and phenylmethanamine (0.058 g, 0.06 mL,
0.54 mmol) were dissolved in acetonitrile (10.0 mL). Triethylamine
(5 equiv, 0.38 mL) was added to the solution, and the reaction mix-
ture stirred at 60 °C for 24 h. Solvent was removed from the reac-
tion by rotary evaporation. The resulting yellow oil was purified
using column chromatography from 2% MeOH/CH2Cl2 to afford
N-benzyl-4,5-dibromo-1H-pyrrole-2-carboxamide (0.0978 g, 51%)
as a brown solid, decomp. 125 °C.
1H NMR (Acetone-d6): d 11.76 (s, 1H), 8.03 (br s, 1H), 7.36–7.29
(m, 4H), 7.26–7.21 (m, 1H), 6.96 (s, 1H), 4.56 (d, J = 5.9 Hz, 2H).
13C NMR (Acetone-d6): d 160.0, 140.3, 129.2 (2 Â C), 129.1,
128.4 (2 Â C), 127.8, 113.3, 105.4, 99.5, 43.4.
HRMS (ESI) m/z: calculated for C18H17N2O [M+H]+ 277.1335;
Found 277.1330.
4.2.1.13.
N-(3-(1H-Pyrazol-1-yl)propyl)-1H-pyrrole-2-carbox-
HRMS (ESI) m/z: calculated for C12H9Br2N2O [MÀH]À 354.9087;
Found 354.9094.
amide (4m). Compound 4m was synthesized according to general
procedure 1 from 2 (0.204 g, 0.94 mmol) and 3-(1H-pyrazol-1-
yl)propan-1-amine (0.118 g, 0.12 mL, 0.94 mmol). The resulting
pale pink oil was purified using column chromatography from 5%
MeOH/CH2Cl2 to afford 4m (0.137 g, 65%) as a white solid, mp
164–166 °C.
4.2.2.2. 4,5-Dibromo-N-phenethyl-1H-pyrrole-2-carboxamide
(5b). Compound 5b was synthesized according to general proce-
dure 2 from 3 (0.200 g, 0.54 mmol) and 2-phenylethylamine
(0.066 g, 0.07 mL, 0.54 mmol). The resulting clear yellow oil was
purified using column chromatography from 2% MeOH/CH2Cl2 to
afford 5b (0.108 g, 53%) as a light yellow solid, mp 156–158 °C.
1H NMR (DMSO-d6): d 12.65 (s, 1H), 8.20 (t, J = 5.4 Hz, 1H),
7.31–7.26 (m, 2H), 7.24–7.17 (m, 3H), 6.89 (s, 1H), 3.42 (q,
J = 6.7 Hz, 2H), 2.80 (t, J = 7.3 Hz, 2H).
1H NMR (DMSO-d6): d 11.42 (s, 1H), 8.02 (t, J = 5.5 Hz, 1H), 7.66
(s, 1H), 7.21 (s, 1H), 6.89 (s, 1H), 6.84 (br s, 1H), 6.75 (br s, 1H),
6.11–6.05 (m, 1H), 4.00 (t, J = 6.9 Hz, 2H), 3.18 (q, J = 6.4 Hz, 2H),
1.92 (p, J = 6.8 Hz, 2H).
13C NMR (DMSO-d6): d 160.8, 137.3, 128.4, 126.2, 121.2, 119.4,
109.8, 108.5, 43.8, 35.7, 31.1.
13C NMR (DMSO-d6): d 158.8, 139.4, 128.6 (2 Â C), 128.3 (2 Â C),
128.3, 126.1, 112.4, 104.4, 97.7, 35.2, 30.7.
HRMS (ESI) m/z: calculated for C11H15N4O [M+H]+ 219.1140;
Found 219.1132.
HRMS (ESI) m/z: calculated for C13H11Br2N2O [M+H]+ 368.9244;
Found 368.9250.
4.2.1.14. N-(3-(2-Methyl-1H-imidazol-1-yl)propyl)-1H-pyrrole-
2-carboxamide (4n). Compound 4n was synthesized according
to general procedure 1 from 2 (0.201 g, 0.94 mmol) and 3-(2-
4.2.2.3. 4,5-Dibromo-N-(3-phenylpropyl)-1H-pyrrole-2-carbox-
amide (5c). Compound 5c was synthesized according to general
procedure 2 from 3 (0.200 g, 0.54 mmol) and 3-phenylpropan-1-
amine (0.073 g, 0.08 mL, 0.54 mmol). The resulting orange oil
was purified using column chromatography from 2% MeOH/CH2Cl2
to afford 5c (0.103 g, 50%) as a yellow solid, mp 115–117 °C.
1H NMR (Acetone-d6): d 12.10 (s, 1H), 7.72 (br s, 1H), 7.30–7.18
(m, 4H), 7.18–7.13 (m, 1H), 6.91 (s, 1H), 3.43 (q, J = 6.7 Hz, 2H),
2.68 (t, J = 7.7 Hz, 2H), 1.91 (quin, J = 7.4 Hz, 2H).
methyl-1H-imidazol-1-yl)propan-1-amine
(0.131 g,
0.13 mL,
0.94 mmol). The resulting white solid was purified using column
chromatography from 2% MeOH/CH2Cl2 to afford 4n (0.110 g,
50%) as a white solid, mp 191–194 °C.
1H NMR (DMSO-d6): d 11.43 (s, 1H), 8.01 (t, J = 5.5 Hz, 1H), 7.09
(s, 1H), 6.84 (br s, 1H), 6.75 (br s, 1H), 6.72 (s, 1H), 6.10–6.04 (m,
1H), 3.89 (t, J = 7.0 Hz, 2H), 3.20 (q, J = 6.4 Hz, 2H), 2.25 (s, 3H),
1.88 (p, J = 6.9 Hz, 2H).
13C NMR (Acetone-d6): d 160.3, 142.7, 129.2 (2 Â C), 129.1
(3 Â C), 126.6, 113.2, 105.4, 99.4, 39.8, 33.8, 32.2.
13C NMR (DMSO-d6): d 160.8, 143.6, 126.2, 126.2, 121.2, 119.6,
109.7, 108.5, 42.9, 35.7, 30.5, 12.6.
HRMS (ESI) m/z: calculated for C14H13Br2N2O [MÀH]À
382.9400; Found 382.9407.
HRMS (ESI) m/z: calculated for C12H17N4O [M+H]+ 233.1397;
Found 233.1400.
4.2.1.15.
amide (4o).
N-(2-(1H-Imidazol-4-yl)ethyl)-1H-pyrrole-2-carbox-
2,2,2-Trichloro-1-(1H-pyrrol-2-yl)ethanone (2)
4.2.2.4. 4,5-Dibromo-N-(4-phenylbutyl)-1H-pyrrole-2-carbox-
amide (5d). Compound 5d was synthesized according to general
procedure 2 from 3 (0.200 g, 0.54 mmol) and 4-phenylbutylamine
(0.081 g, 0.09 mL, 0.54 mmol). The resulting yellow oil was purified
using column chromatography from 50% EtOAc/Hexane to afford
5d (0.136 g, 63%) as an off-white solid, mp 126–128 °C.
1H NMR (DMSO-d6): d 12.64 (s, 1H), 8.09 (br s, 1H), 7.30–7.22
(m, 2H), 7.22–7.12 (m, 3H), 6.90 (s, 1H), 3.28–3.15 (m, 2H), 2.58
(t, J = 7.2 Hz, 2H), 1.63–1.53 (m, 2H), 1.53–1.44 (m, 2H).
13C NMR (DMSO-d6): d 158.7, 142.1, 128.3 (3 Â C), 128.2 (2 Â C),
125.6, 112.3, 104.2, 97.7, 38.3, 34.8, 28.9, 28.4.
(0.277 g, 1.30 mmol) and histamine dihydrochloride (0.240 g,
1.30 mmol) were dissolved in N,N-dimethylformamide (4.0 mL).
Piperidine (5 equiv, 0.27 mL) was added to the solution and the
reaction mixture was subjected to microwave irradiation at
150 °C for 5 min. Solvent was removed from the reaction by rotary
evaporation. The resulting yellow oil was triturated from hot eth-
anol to afford 4o (0.032 g, 12%) as a white solid, mp 212–214 °C.
1H NMR (DMSO-d6): d 11.80 (br s, 1H), 11.39 (s, 1H), 8.06 (t,
J = 5.4 Hz, 1H), 7.52 (s, 1H), 6.90–6.74 (m, 2H), 6.72 (br s, 1H),
6.06 (dd, J = 5.4, 2.3 Hz, 1H), 3.42 (q, J = 6.9 Hz, 2H), 2.72 (t,
J = 7.3 Hz, 2H).
HRMS (ESI) m/z: calculated for C15H15Br2N2O [MÀH]À
396.9557; Found 396.9559.

1698
L. Dyson et al. / Bioorg. Med. Chem. 22 (2014) 1690–1699
4.2.2.5. 4,5-Dibromo-N-(4-chlorobenzyl)-1H-pyrrole-2-carbox-
amide (5e). Compound 5e was synthesized according to general
procedure 2 from 3 (0.200 g, 0.54 mmol) and (4-chlorophenyl)
methanamine (0.077 g, 0.07 mL, 0.54 mmol). The resulting clear
oil was purified using column chromatography from 2% MeOH/
CH2Cl2 to afford 5e (0.080 g, 37%) as an off-white solid, mp
202–204 °C.
1H NMR (DMSO-d6): d 12.65 (s, 1H), 8.19 (t, J = 5.6 Hz, 1H), 7.12
(s, 4H), 6.89 (s, 1H), 3.40 (q, J = 6.8 Hz, 2H), 2.76 (t, J = 7.3 Hz, 2H),
2.55 (q, J = 7.6 Hz, 2H), 1.15 (t, J = 7.6 Hz, 3H).
13C NMR (DMSO-d6): d 158.8, 141.4, 136.6, 128.6 (2 Â C), 128.3,
127.7 (2 Â C), 112.4, 104.4, 97.8, 40.3, 34.8, 27.8, 15.6.
HRMS (ESI) m/z: calculated for C15H15Br2N2O [MÀH]À
396.9557; Found 396.9566.
1H NMR (DMSO-d6): d 12.72 (s, 1H), 8.69 (t, J = 5.9 Hz, 1H), 7.38
(d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H), 6.97 (s, 1H), 4.41 (d,
J = 5.9 Hz, 2H).
4.2.2.10. 4,5-Dibromo-N-(2-(naphthalen-2-yl)ethyl)-1H-pyrrole-
2-carboxamide (5j). Compound 5j was synthesized according to
general procedure 2 from 3 (0.202 g, 0.54 mmol) and N-methyl-2-
(naphthalen-2-yl)ethanamine (0.092 g, 0.54 mmol). The resulting
orange oil was purified using column chromatography from 50%
EtOAc/Hexane to afford 5j (0.101 g, 44%) as a yellow solid, mp
166–169 °C.
13C NMR (DMSO-d6): d 158.9, 138.6, 131.3, 129.0 (2 Â C), 128.2
(2 Â C), 127.9, 112.8, 104.8, 97.9, 41.3.
HRMS (ESI) m/z: calculated for C12H8Br2N2O [MÀH]À 388.8697;
Found 388.8702.
4.2.2.6. 4,5-Dibromo-N-(3,4-dichlorobenzyl)-1H-pyrrole-2-car-
boxamide (5f). Compound 5f was synthesized according to
general procedure 2 from 3 (0.200 g, 0.54 mmol) and 3,4-dichloro-
benzylamine (0.095 g, 0.07 mL, 0.54 mmol). The resulting yellow
oil was purified using column chromatography from 1% MeOH/
CH2Cl2 to afford 5f (0.113 g, 49%) as an off-white solid, mp
212–214 °C.
1H NMR (DMSO-d6): d 12.65 (s, 1H), 8.24 (t, J = 5.6 Hz, 1H),
7.89–7.81 (m, 3H), 7.73 (s, 1H), 7.51–7.39 (m, 3H), 6.89 (d,
J = 2.0 Hz, 1H), 3.54 (q, J = 6.7 Hz, 2H), 2.97 (t, J = 7.2 Hz, 2H).
13C NMR (DMSO-d6): d 158.8, 137.1, 133.1, 131.7, 128.2, 127.7,
127.5, 127.5, 127.3, 126.7, 126.0, 125.3, 112.4, 104.4, 97.7, 40.1,
35.4.
HRMS (ESI) m/z: calculated for C17H13Br2N2O [MÀH]À
418.9400; Found 418.9408.
1H NMR (DMSO-d6): d 12.75 (s, 1H), 8.72 (t, J = 5.9 Hz, 1H), 7.59
(d, J = 8.3 Hz, 1H), 7.54 (d, J = 1.2 Hz, 1H), 7.28 (dd, J = 8.2, 1.3 Hz,
1H), 6.97 (s, 1H), 4.41 (d, J = 6.0 Hz, 2H).
4.2.2.11. 4,5-Dibromo-N-(2,2-diphenylethyl)-1H-pyrrole-2-car-
boxamide (5k). Compound 5k was synthesized according to
general procedure 2 from 3 (0.200 g, 0.54 mmol) and 2,2-dipheny-
lethanamine (0.110 g, 0.54 mmol). The resulting white solid was
purified using column chromatography from 1% MeOH/CH2Cl2 to
afford 5k (0.117 g, 48%) as a white solid, 199-202 °C.
1H NMR (DMSO-d6): d 12.59 (s, 1H), 8.16 (br s, 1H), 7.39–7.23
(m, 8H), 7.22–7.13 (s, 2H), 6.80 (s, 1H), 4.32 (t, J = 7.4 Hz, 1H),
3.94–3.77 (m, 2H).
13C NMR (DMSO-d6): d 159.0, 140.8, 130.9, 130.5, 129.3, 129.2,
127.8, 127.6, 112.9, 105.0, 97.9, 41.0.
HRMS (ESI) m/z: calculated for C12H7Br2Cl2N2O [MÀH]À
422.8308; Found 422.8317.
4.2.2.7.
4,5-Dibromo-N-(4-methoxybenzyl)-1H-pyrrole-2-car-
boxamide (5g). Compound 5g was synthesized according to
general procedure 2 from 3 (0.213 g, 0.57 mmol) and 4-methoxy-
benzylamine (0.079 g, 0.08 mL, 0.57 mmol). The resulting yellow
oil was triturated from hot ethanol to afford 5g (0.078 g, 35%) as
a beige solid, mp 194–196 °C.
13C NMR (DMSO-d6): d 159.2, 143.0 (2 Â C), 128.8 (4 Â C), 128.2
(5 Â C), 126.8 (2 Â C), 112.9, 104.9, 98.2, 50.4, 43.4.
HRMS (ESI) m/z: calculated for C19H15Br2N2O [MÀH]À
444.9557; Found 444.9565.
1H NMR (DMSO-d6): d 12.69 (s, 1H), 8.58 (t, J = 5.9 Hz, 1H), 7.21
(d, J = 8.6 Hz, 2H), 6.96 (s, 1H), 6.88 (d, J = 8.6 Hz, 2H), 4.35 (d,
J = 5.9 Hz, 2H), 3.72 (s, 3H).
4.2.2.12. N-(Biphenyl-4-ylmethyl)-4,5-dibromo-1H-pyrrole-2-
carboxamide (5l). Compound 5l was synthesized according to
general procedure 2 from 3 (0.200 g, 0.54 mmol) and biphenyl-4-
ylmethanamine (0.099 g, 0.54 mmol). The resulting yellow solid
was purified using column chromatography from 1% MeOH/CH2Cl2
to afford 5l (0.099 g, 44%) as an off-white solid, mp136–139 °C.
1H NMR (DMSO-d6): d 12.73 (s, 1H), 8.70 (t, J = 6.0 Hz, 1H),
7.67–7.59 (m, 4H), 7.49–7.42 (m, 2H), 7.41–7.32 (m, 3H), 7.00 (d,
J = 2.3 Hz, 1H), 4.47 (d, J = 6.0 Hz, 2H).
13C NMR (DMSO-d6): d 158.8, 158.2, 131.4, 128.6 (2 Â C), 128.1,
113.7 (2 Â C), 112.6, 104.6, 97.8, 55.1, 41.4.
HRMS (ESI) m/z: calculated for C13H11Br2N2O2 [MÀH]À
384.9193; Found 384.9198.
4.2.2.8.
4,5-Dibromo-N-(4-hydroxyphenethyl)-1H-pyrrole-2-
carboxamide (5h). Compound 5h was synthesized according
to general procedure 2 from 3 (0.203 g, 0.54 mmol) and 4-(2-ami-
noethyl)phenol (0.074 g, 0.54 mmol). The resulting brown oil was
purified using column chromatography from 2% MeOH/CH2Cl2 to
afford 5h (0.114 g, 53%) as a light brown solid, mp 167–169 °C.
1H NMR (DMSO-d6): d 12.63 (s, 1H), 9.16 (s, 1H), 8.15 (br s, 1H),
7.00 (d, J = 6.0 Hz, 2H), 6.89 (s, 1H), 6.67 (d, J = 6.4 Hz, 2H), 3.34 (br
s, 2H), 2.67 (br s, 2H).
13C NMR (DMSO-d6): d 158.9, 139.9, 138.8, 138.7, 128.9 (2 Â C),
128.0, 127.8 (2 Â C), 127.3, 126.6 (4 Â C), 126.6, 112.7, 104.7, 97.9,
41.7.
HRMS (ESI) m/z: calculated for C18H13Br2N2O [MÀH]À
430.9400; Found 430.9407.
13C NMR (DMSO-d6): d 158.7, 155.6, 129.5 (2 Â C), 129.4, 128.3,
115.1 (2 Â C), 112.4, 104.4, 97.7, 40.6, 34.4.
4.2.2.13.
N-(3-(1H-Pyrazol-1-yl)propyl)-4,5-dibromo-1H-pyr-
role-2-carboxamide (5m). Compound 5m was synthesized
according to general procedure 2 from 3 (0.207 g, 0.54 mmol)
HRMS (ESI) m/z: calculated for C13H11Br2N2O2 [MÀH]À
384.9193; Found 384.9195.
and
3-(1H-pyrazol-1-yl)propan-1-amine
(0.068 g,
0.07 mL,
0.54 mmol). The resulting white solid was purified using column
chromatography from 50% EtOAc/Hexane to afford 5m (0.134 g,
64%) as a light brown solid, decomp. 202 °C.
4.2.2.9.
4,5-Dibromo-N-(4-ethylphenethyl)-1H-pyrrole-2-car-
boxamide (5i). Compound 5i was synthesized according to
general procedure 2 from 3 (0.202 g, 0.54 mmol) and (4-ethylphe-
nyl)methanamine (0.073 g, 0.09 mL, 0.54 mmol). The resulting
yellow oil was purified using column chromatography from 2%
MeOH/ CH2Cl2 to afford 5i (0.064 g, 31%) as a yellow solid, mp
166–167 °C.
1H NMR (DMSO-d6): d 12.49 (s, 1H), 8.14 (br s, 1H), 7.65 (s, 1H),
7.19 (s, 1H), 6.92 (s, 1H), 6.89 (s, 1H), 3.99 (t, J = 6.5 Hz, 2H), 3.21–
3.14 (m, 2H), 1.96–1.86 (m, 2H).
13C NMR (DMSO-d6): d 159.0, 137.3, 128.4, 128.2, 119.3, 112.5,
104.5, 97.7, 43.7, 35.8, 30.8.

L. Dyson et al. / Bioorg. Med. Chem. 22 (2014) 1690–1699
1699
HRMS (ESI) m/z: calculated for C11H11Br2N4O [MÀH]À
13. Wright, A. D.; König, G. M.; Angerhofer, C. K.; Greenidge, P.; Linden, A.;
Desqueyroux-Faundez, R. J. Nat. Prod. 1996, 59, 710.
14. König, G. M.; Wright, A. D.; Franzblau, S. G. Planta Med. 2000, 66, 337.
15. Mayer, A. M. S.; Glaser, K. B.; Cuevas, C.; Jacobs, R. S.; Kem, W.; Little, R. D.;
McIntosh, J. M.; Newman, D. P.; Potts, B. C.; Shuster, D. E. Trend Pharm. Sci.
2010, 31, 255.
16. Harvey, A. L. Drug Discovery Today 2008, 13, 894.
17. Holland, I. P.; McCluskey, A.; Sakoff, J. A.; Chau, N.; Robinson, P. J.; Motti, C. A.;
Wright, A. D.; van Altena, I. A. J. Nat. Prod. 2009, 72, 102.
372.9305; Found 372.9309.
4.2.2.14.
pyl)-1H-pyrrole-2-carboxamide (5n). Compound 5n was syn-
thesized according to general procedure from (0.204 g,
4,5-Dibromo-N-(3-(2-methyl-1H-imidazol-1-yl)pro-
2
3
0.54 mmol) and 3-(2-methyl-1H-imidazol-1-yl)propan-1-amine
(0.075 g, 0.07 mL, 0.54 mmol). The resulting brown solid was puri-
fied using column chromatography from 5% MeOH/CH2Cl2 to afford
5n (0.027 g, 13%) as a white solid, decomp. 219 °C.
1H NMR (DMSO-d6): d 8.17 (br s, 1H), 7.10 (s, 1H), 6.92 (s, 1H),
6.75 (s, 1H), 3.90 (t, J = 7.0 Hz, 2H), 3.19 (q, J = 6.4 Hz, 2H), 2.26 (s,
3H), 1.87 (p, J = 6.8 Hz, 2H).
18. Wright, A. D.; McCluskey, A.; Robertson, M. J.; MacGregor, K.; Gordon, C. P.;
Guenther, J. Org. Biomol. Chem. 2011, 9, 400.
19. Forenza, S.; Minale, L.; Riccio, R.; Fattorusso, E. J. Chem. Soc. 1971, 1129.
20. Garcia, E. E.; Benjamin, L. E.; Fryer, R. I. J. Chem. Soc., Chem. Commun. 1973, 78.
21. van Soest, R. Agelas oroides (Schmidt, 1864) 2007. Available from: http://
www.marinespecies.org/aphia.php?p=taxdetails&id=132454.
22. Scala, F.; Fattorusso, E.; Menna, M.; Taglialatela-Scafati, O.; Tierney, M.; Kaiser,
M.; Tasdemir, D. Mar. Drugs 2010, 8, 2162.
23. Forte, B.; Malgesini, B.; Piutti, C.; Quartieri, F.; Scolaro, A.; Papeo, G. Mar. Drugs
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Cavanagh, J.; Melander, C. J. Med. Chem. 2009, 52, 4582.
13C NMR (DMSO-d6): d 159.1, 143.6, 128.2, 125.9, 119.7, 112.6,
104.6, 97.8, 42.9, 35.9, 30.2, 12.5.
HRMS (ESI) m/z: calculated for C12H13Br2N4O [MÀH]À
386.9462; Found 386.9470.
25. McCluskey, A.; Bowyer, M. C.; Collins, E.; Sim, A. T. R.; Sakoff, J. A.; Baldwin, M.
L. Bioorg. Med. Chem. Lett. 2000, 10, 1687.
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M. A.; Walkom, C.; Sakoff, J. A. Anticancer Drug Des. 2001, 16, 291.
27. 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.
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A.; Robinson, P. J. Mol. Cancer Ther. 2011, 10, 1553.
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M. J.; MacGregor, K. A.; Tomlin, N.; Pechstein, A.; Chau, N.; Chircop, M.; Sakoff,
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4.2.3. General procedure 3
4.2.3.1. N-(2-(1H-Imidazol-4-yl)ethyl)-4,5-dibromo-1H-pyrrole-
2-carboxamide (5o). 2,2,2-Trichloro-1-(4,5-dibromo-1H-pyrrol-
2-yl)ethanone (3) (0.204 g, 0.54 mmol and histamine dihydrochlo
ride (0.150 g, 0.81 mmol) were dissolved in N,N-dimethylformamide
(4.0 mL). Piperidine (5 equiv, 0.27 mL) was added to the solution
and the reaction mixture was subjected to microwave irradiation
at 150 °C for 5 min. Solvent was removed from the reaction by
rotary evaporation. The resulting light brown oil was purified using
column chromatography from 5% MeOH/CH2Cl2 to afford N-(2-
(1H-imidazol-4-yl)ethyl)-4,5-dibromo-1H-pyrrole-2-carboxamide
(0.076 g, 38%) as a light brown solid, decomp. 207 °C.
1H NMR (DMSO-d6): d 8.18 (t, J = 5.5 Hz, 1H), 7.53 (s, 1H), 6.89
(s, 1H), 6.80 (s, 1H), 3.42 (q, J = 6.8 Hz, 2H, obscured by water sig-
nal), 2.71 (t, J = 7.4 Hz, 2H).
13C NMR (DMSO-d6): d 158.8, 134.7 (2 Â C), 128.3, 116.7 (br),
112.4, 104.4, 97.7, 38.8, 27.1.
HRMS (ESI) m/z: calculated for C10H9Br2N4O [MÀH]À 358.9149;
Found 358.9155.
36. Smith, C. M.; Hauke, V.; McCluskey, A.; Robinson, P. J.; Chircop, M. Mol. Cancer
2013, 12. http://dx.doi.org/10.1186/1476-4598-12-4.
37. Deane, F. M.; Maguire, A. R.; Sakoff, J. A.; McCluskey, A.; McCarthy, F. O. Org.
Biomol. Chem. 2013, 11, 1334.
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Yousufuddin, M. Heterocycles 2010, 80, 1353.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.01.021.
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