Synthesis and evaluation of novel anti-proliferative pyrroloazepinone and indoloazepinone oximes derived from the marine natural product hymenialdisine

Article abstract of DOI:10.1016/j.ejmech.2012.08.022

The tetrahydroazepinone pharmacophore is a component of many interesting compounds, including several marine natural products, with anti-cancer properties. The synthesis and biological evaluation of a novel series of pyrroloazepinone and indoloazepinone oximes is reported. These compounds showed promising growth inhibition activity against four human cancer cell lines but did not significantly inhibit the cell cycle regulator cyclin dependent kinase 2. The most active compounds in this series displayed improved anti-proliferative activity over the related synthetic indoloazepine kenpaullone. The structure activity relationships exhibited by the azepinone pharmacophore suggests several novel lead compounds for anti-cancer drug discovery.

Full text of DOI:10.1016/j.ejmech.2012.08.022

European Journal of Medicinal Chemistry 56 (2012) 246e253
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of novel anti-proliferative pyrroloazepinone and
indoloazepinone oximes derived from the marine natural product hymenialdisine
*
Alex W. White , Nicholas Carpenter, Jerome R.P. Lottin, Richard A. McClelland, Robert I. Nicholson
School of Pharmacy and Pharmaceutical Science, Cardiff University, King Edward VII Avenue, Cardiff CF10 3NB, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The tetrahydroazepinone pharmacophore is a component of many interesting compounds, including
several marine natural products, with anti-cancer properties. The synthesis and biological evaluation of
a novel series of pyrroloazepinone and indoloazepinone oximes is reported. These compounds showed
promising growth inhibition activity against four human cancer cell lines but did not significantly inhibit
the cell cycle regulator cyclin dependent kinase 2. The most active compounds in this series displayed
improved anti-proliferative activity over the related synthetic indoloazepine kenpaullone. The structure
activity relationships exhibited by the azepinone pharmacophore suggests several novel lead compounds
for anti-cancer drug discovery.
Received 17 May 2012
Received in revised form
3 August 2012
Accepted 16 August 2012
Available online 24 August 2012
Keywords:
Tetrahydroazepinone
Hymenialdisine
Kenpaullone
Crown Copyright Ó 2012 Published by Elsevier Masson SAS. All rights reserved.
Marine natural products
Oximes
1. Introduction
dependent kinase 1 (CDK1/cyclin B IC50 ¼ 22 nM) and CDK2
(CDK2/cyclin E IC50 ¼ 40 nM) [9]. Its potent inhibition of glycogen
Marine organisms are an important source of natural products
with therapeutic potential, in particular marine sponges have
yielded many compounds with interesting anti-cancer activity
[1,2]. Several prominent examples of marine-derived drugs have
been licenced in recent years. Eribulin, a semi-synthetic macro-
lactone derivative of halichondrin B, has recently been approved in
several countries for the treatment of taxane and anthracycline
resistant metastatic breast cancer [3]. Halichondrin B is a marine
natural product that was originally isolated from the marine
sponge Halichondria okadai but was subsequently found in other,
unrelated, species of sponge [4]. Trabectedin is a complex alkaloid
isolated from the marine tunicate Ecteinascidia turbinata. It was
licenced in Europe in 2007 as a second line treatment for soft tissue
sarcomas following failure of anthracyclines and ifosfamide and
later obtained additional approval in combination with pegylated
liposomal doxorubicin for refractory platinum-sensitive ovarian
cancer [5].
synthase kinase 3
b
(GSK3
b
, IC50 ¼ 10 nM) highlights potential
applications for treating neurodegenerative disorders [9,10]. The
close analogue debromohymenialdisine (also a marine natural
product) has been reported to be a good inhibitor of the cell cycle
regulator checkpoint kinase 2 (Chk2, IC50 ¼ 42 nM) [11]. The fused
heterocyclic azepinone pharmacophore exemplified within
hymenialdisine is also present in the synthetic indoloazepinone
kenpaullone (2) [12]. Kenpaullone belongs to the paullone family
of synthetic indoloazepine kinase inhibitors and is a good inhib-
itor of CDK1/cyclin
B
(IC50
¼
0.4 mM) and CDK2/cyclin A
(IC50 ¼ 0.68 M) [12,13]. The total synthesis of hymenialdisine has
m
been reported [14,15] and structure activity relationship studies
have been undertaken to explore the medicinal chemistry of the
azepinone scaffold employing 1 and 2 as lead compounds. For
example, aminoalkyl extensions (Fig. 1, 3) to the paullone scaffold
were added to probe the active site of CDK1 [16]. An indole
derivative of hymenialdisine (4) has been reported to inhibit
checkpoint kinase 2 (IC50 ¼ 8 nM), and the production of
Hymenialdisine (1), is a bromopyrrole alkaloid [6] isolated
from a variety of marine sponges including Hymeniacidon species
[7,8]. It has been shown to be a potent inhibitor of cyclin
interleukin-2 (IC50 ¼ 3.5
to exhibiting excellent growth inhibition properties against
M) that are comparable to the
m
M) and TNF
a
(IC50 ¼ 8.2
mM), in addition
leukaemia T-cells (GI50 ¼ 1.7
m
parent natural product [11,17]. A series of hydrazone derivatives in
both the pyrrole and indole series (Fig. 1, 5) are reported to show
improved anti-proliferative activity relative to hymenialdisine
* Corresponding author. Tel.: þ44 (0)29 2087 6308; fax: þ44 (0) 2920 874149.
E-mail address: whiteaw@cf.ac.uk (A.W. White).
0223-5234/$ e see front matter Crown Copyright Ó 2012 Published by Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.08.022

A.W. White et al. / European Journal of Medicinal Chemistry 56 (2012) 246e253
247
over the polyphosphoric acid method (54% versus 49%) but proved
significantly easier to perform [20]. Synthesis of the unsubstituted
oxime 9a proceeded readily by treatment of 8 with hydroxylamine
hydrochloride and sodium acetate in aqueous ethanol. Further
substitution of the oxime functional group to extend the pharma-
cophore required basic conditions necessitating a protecting group
strategy for the pyrrole-NH which proved highly problematic. The
direct reaction of commercially available O-benzylhydroxylamine
with ketone 8 yielded the desired substituted oxime 9b in an
acceptable yield (27%). The E-stereochemistry of this and all further
reported oximes was established by 2D-NMR NOESY experiments.
The E-oximes were isolated exclusively with no evidence of the Z-
stereoisomers.
The presence of a bromine atom in both hymenialdisine (1) and
kenpaullone (2) provided a strong rationale for the synthesis of
brominated analogues. Mono-bromination of the pyrrole was
attempted but resulted in an inseparable mixture of isomers.
However, starting material 6 was readily converted to the dibromo
derivative 6a by treatment with bromine at 0 ꢀC in good yield (70%).
Starting from 6a, the above synthesis was readily applied to prepare
the dibromopyrroloazepinone oxime 9c.
Fig. 1. The marine natural product hymenialdisine (1) and synthetic analogues con-
taining the pyrrolo- and indoloazepinone pharmacophore.
[18]. Herein we report the synthesis and evaluation of novel oxime
derivatives of the pyrrolo- and indoloazepinone scaffold of 1 and
2. The use of an oxime-linker to extend the azepinone pharma-
cophore is of interest as a convenient, non-chiral, functional group
interconversion from a carbonyl group, and as a probe for struc-
ture activity relationships beyond the chemical space occupied,
for example, by the cyclic guanidinone group of hymenialdisine
(1).
The synthesis of the N-methylpyrrole derivatives (Scheme 2)
utilised a slightly modified synthesis starting with commercially
available carboxylic acid 10. Efficient coupling with
b-alanine
ethyl ester using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
(EDCI) followed by basic ester hydrolysis yielded cyclisation
precursor 12 which readily reacted with P2O5 and methanesulfonic
acid to yield pyrroloazepine 12 in excellent yield (73% over three
steps). Oxime 9d was obtained in 82% yield from 12. The blocking
N-methyl group permitted ready substitution of the oxime by
treatment with a strong base and benzyl bromide or (bromo-
methyl) cyclohexane to yield target oximes 9f and 9h respectively
in good yield.
2. Chemistry
2.1. Pyrroloazepinone synthesis
The synthesis of brominated N-methylpyrroles was initially
attempted as outlined above. N-Methylpyrrole 10 was readily
dibrominated by treatment with bromine at 0 ꢀC (73% yield) but
debromination unexpectedly occurred in the subsequent cyclisa-
tion step. This problem was avoided by conversion of cyclised
derivative 9d to the dibromo-derivative 9e (Scheme 2), which was
used as the precursor for the synthesis of substituted oximes 9g
and 9i.
Many previously reported pyrroloazepinone syntheses proceed
by condensation of b-alanine with pyrrole derivatives [13,16,19]. As
outlined in Scheme 1, commercially available 2-trichloroacetyl-
pyrrole (6) is a commonly used starting material. Amide forma-
tion in good yield (63%) under mild conditions was followed by
basic ester hydrolysis to yield the carboxylic acid 7 essential for the
key cyclisation step to pyrroloazepinone 8. The cyclisation proved
to be the limiting step in this synthesis and several methods were
investigated. Polyphosphoric acid was used successfully, but an
alternative procedure was identified using phosphorus pentoxide
and methanesulfonic acid that gave a slight improvement in yield
H
OH
N
OH
O
a,b
N
CH3
10
N
CH3
O
O
11
R
R
OH
O
HN
O
R=H
7a R=Br
OH
O
CCl3
O
a,b
N
R
R
N
H
N
H
R
O
c
d
NH
6
R=H
7
NH
f
f
N
R
N
6a R=Br
O
CH3
CH3
OR1
O
N
12
9d R=H
9e R=Br
R
R
R
c
d or e
OR1
NH
N
NH
N
R
N
R
9f R=H R1=Bn
9g R=Br R1=Bn
9h R=H R1=CH2C6H11
9i R=Br R1=CH2C6H11
H
O
H
O
e
8
R=H
8a R=Br
9a R=H, R1=H
9b R=H, R1=CH2Ph
9c R=Br,R1=CH2Ph
NH
R
N
CH3
O
Scheme 1. Reagents and conditions: (a)
b
-Alanine ethyl ester, diisopropylamine, THF,
Scheme 2. Reagents and conditions: (a) b-Alanine ethyl ester, EDCI, DMAP, dichloro-
reflux; (b) LiOH.H2O, THF; (c) MeSO3H, P2O5, 80 ꢀC; (d) NH2OH, AcONa.3H2O, EtOH,
methane; (b) LiOH.H2O, THF; (c) MeSO3H, P2O5, 80 ꢀC; (d) NH2OH, AcONa.3H2O, EtOH,
H2O, 60 ꢀC; (e) KOtBu, R1Br, THF; (f) Br2, THF, 0 ꢀC.
H2O, 60 ꢀC; (e) BnONH2, AcOH; (f) Br2, THF, 0 ꢀC.

248
A.W. White et al. / European Journal of Medicinal Chemistry 56 (2012) 246e253
with b-alanine ethyl ester using a carbodiimide coupling reagent
a (18),
b (23),
c
was achieved in high yield (85%) with no need for N-protection.
Subsequent basic hydrolysis yielded the cyclisation precursor 14.
The cyclodehydration to indoloazepinone 15 was achieved in 58%
OH
O
HN
O
OH
O
N
R
N
R
yield using methanesulfonic acid
e phosphorous pentoxide.
Reagents and conditions used for pyrrolo-oxime synthesis proved
unsuccessful, but addition of a Lewis acid catalyst (BF3.OEt2)
provided a route to the indoloazepinone oxime derivatives 16a and
16b. The N-methyl indole derivatives were synthesized from
13 R=H
14 R=H
17 R=CH3
18 R=CH3
e (20,21),
OR1
O
f (25),
N
compound 17. In contrast to previous couplings with b-alanine, the
g (26,27)
d
most efficient method for the preparation of 18 proved to be via
acid chloride activation of the carboxylic acid group of 17 (see
Scheme 3). The cyclisation to 19 mediated by methanesulfonic acid
and phosphorous pentoxide proceeded in excellent yield (82%).
Functional group interconversion of the ketone to oximes 16c, 16d
and 16e proceeded by the established methods. The synthesis of
a brominated indole analogue was readily achieved by treatment
of 15 with N-bromosuccinamide (NBS). Monobrominated deriva-
tive 20 was isolated in high yield (88%) and converted to the
oxime 16f by treatment with hydroxylamine in the presence of
a Lewis acid.
In general, the synthesis of the N-methyl derivatives in both the
pyrrolo- and indoloazepinone series occurred more readily than
the preparation of the free NH analogues. Therefore, the incor-
poration of pyrrole/indole nitrogen protecting groups at various
stages of synthesis was investigated with the aim of establishing
a generic protection strategy to assist the preparation of a greater
selection of analogues. Despite investigating a variety of chemis-
tries (for example: N-benzyl, N-tosyl, various N-silyl) the methods
reported within this paper proved by far the most concise, effi-
cient and high yielding for the synthesis of this series of
compounds.
NH
NH
N
R
N
R
O
O
16a R=H R1=H
15 R=H
19 R=CH3
16b R=H R1=CH2Ph
16c R=CH3 R1=H
16d R=CH3 R1=CH2Ph
16e R=CH3 R1=CH2C6H11
h
Br
R
20 R=O
e
NH
16f R=NOH
N
H
O
Scheme 3. Reagents and conditions: (a) b-Alanine ethyl ester, EDCI, DMAP, dichloro-
methane; (b) (COCl)2, TEA, DMF, THF; (c) LiOH.H2O, THF; (d) MeSO3H, P2O5, 80 ꢀC; (e)
NH2OR, BF3.OEt2, pyridine, THF; (f) NH2OH, AcONa.3H2O, EtOH, H2O, 60 ꢀC; (g) KOtBu,
RBr, THF; (h) NBS, DMF.
2.2. Indoloazepinone synthesis
The synthesis of the indoloazepinone series was achieved as
outlined in Scheme 3. Coupling of indole-2-carboxylic acid (13)
Table 1
Biological evaluation of pyrroloazepinones A and indoloazepinones B.
Structure
R
R1
R2
GI50
(
m
M)a
CDK2% Inhibitionb
A549
LoVo
MCF7
PC3
8
8a
A
A
A
B
B
A
A
B
B
B
A
A
B
A
A
B
A
A
B
H
H
CH3
H
CH3
H
CH3
H
CH3
H
H
H
H
Br
H
H
H
H
H
H
H
Br
H
Br
H
H
Br
H
H
Br
H
O
O
O
O
O
NOH
NOH
NOH
>100
>100
>100
97.7
>100
>100
>100
>100
>100
63.3
>100
>100
>100
36.9
36.8
>100
>100
40.5
51.3
27.2
51.7
23.6
24.3
82.4
77.3
ND
>100
>100
>100
>100
>100
>100
>100
>100
>100
54.6
39.2
26.3
28.1
93.9
66.7
ND
45.8
31.3
>100
>100
>100
>100
>100
>100
>100
>100
>100
46.2
52.7
26.3
23.4
97.0
96.4
ND
44.4
32.7
NTc
NT
NT
NT
NT
NT
NT
NT
NT
45
NT
0
32
NT
22
NT
NT
13
12
12
15
19
9a
9d
16a
16c
16f
9b
9c
16b
9f
9g
16d
9h
9i
NOH
NOH
NOCH2Ph
NOCH2Ph
NOCH2Ph
NOCH2Ph
NOCH2Ph
NOCH2Ph
NOCH2C6H11
NOCH2C6H11
NOCH2C6H11
87.5
40.5
34.9
H
CH3
CH3
CH3
CH3
CH3
CH3
>100
61.1
NDd
74.2
36.4
32.1
48.2
21.6
20.5
16e
23.3
29.1
a
Growth inhibition 50% (concentration required to reduce the growth of cells by 50% relative to an untreated control. Mean of at least three experiments).
CDK2-cyclin A % inhibition at 10 mM.
b
c
NT ¼ Not tested.
d
ND ¼ Not determined e decomposed under assay conditions.

A.W. White et al. / European Journal of Medicinal Chemistry 56 (2012) 246e253
249
3. Biological results and discussion
4. Conclusion
The results obtained from the screening of all compounds
against four human cancer cell lines: A549 (lung), LoVo (colo-
rectal), MCF-7 (breast) and PC3 (prostate) and selected screening
against CDK2-cyclin A, a potential cellular target, is presented in
Table 1.
The pyrroloazepinediones 8, 8a and 12 were inactive across the
cell line panel. By contrast, the analogous indoloazepinediones 15
and 19 were selectively active against the LoVo cell line. Conversion
of the ketone to an unsubstituted oxime showed no improvement
in activity for the pyrrole derivatives 9a and 9d but activity and
selectively was retained for indoles 16a and 16c (compared with 15
and 19 respectively). Incorporation of the bromine substituent in
indole 16f produced a significant increase in activity across all four
cell lines.
Marine sponges continue to yield compounds of interest to drug
discovery, although there are many barriers to their clinical use,
including ease of production/synthesis and suitable pharmaceutical
properties. A series of novel oxime substituted pyrrolo- and indo-
loazepinones inspired by the marine natural product hymenialdi-
sine have been synthesized and shown to exhibit excellent anti-
proliferative properties against a panel of human tumour cell
lines. Against expectations, these compounds were not significant
CDK2/cyclin A inhibitors, but gratifyingly several compounds dis-
played anti-proliferative activity at least comparable to the related
and well characterised compound kenpaullone. In summary, we
present a series of synthetically accessible novel compounds that
are of interest for further development as novel agents for the
treatment of cancer.
Bulky aromatic or aliphatic substitution of the oxime func-
tional group resulted in compounds with an improved response.
Good inhibition of cell proliferation was observed for all these
compounds (9b, 9c, 9f, 9g, 9h, 9i, 16b, 16d and 16e) across all four
5. Experimental
5.1. General protocols
cell lines. Although
a substituted oxime was essential for
increased potency, there was no clear structure activity relation-
ship between the O-benzyl or O-cyclohexylmethyl substitutions.
Similarly, the addition of a bromine substituent, inspired by the
presence of this functional group in many marine natural prod-
ucts and the related synthetic kinase inhibitor kenpaullone (2),
did not offer any clear structure activity relationship. Despite this,
compounds 9c, 9i, 16b and 16e showed improved cell line activity
All solvents were of reagent grade and purchased from Fisher
Scientific UK. All anhydrous solvents were purchased from Lan-
caster or SigmaeAldrich in sure-seal bottles. Water refers to de-
ionized water. All reactions were monitored routinely by thin
layer chromatography (TLC) performed on commercially available
Merck Kieselgel 60F254 plates and visualized using ultraviolet light
(254 nm or 366 nm) or by treatment with iodine or permanganate
dip. Column chromatography was performed in glass columns
slurry packed in the appropriate eluent under gravity using silica
gel 60. Samples were applied as a concentrated solution in the same
eluent, or pre-absorbed onto silica gel. Fractions containing product
were identified by TLC, combined, and the solvent removed under
vacuum. Melting points (mp) were determined in a capillary on an
electric variable heater and are not corrected. Infrared spectra (IR)
were recorded on a Perkin Elmer 1600 series FTIR spectrometer as
solids via a diffuse reflectance accessory using a dry potassium
bromide matrix. 1H and 13C NMR were recorded on a Bruker Avance
DPX500 spectrometer with operating frequencies of 500 MHz and
125 MHz respectively. Spectra were recorded in d6-DMSO unless
stated otherwise. Low resolution electrospray mass spectra (LRMS)
were run on a VG platform II Fisons instrument in either positive or
negative mode using a mobile phase of methanol. High resolution
mass spectrometry (HRMS) was performed by the EPRSC National
Mass Spectrometry Service Centre, Swansea University, UK, using
electron impact/chemical ionization (EI/CI) and electrospray ion-
isation methods. Elemental analyses were performed by Medac
Ltd., Surrey, UK.
in our assay compared against a mean GI50 ¼ 42
mM for ken-
paullone in the NCI human tumour 60-cell line screen [12].
Furthermore, the series of compounds appear to show a selec-
tivity for activity against the colorectal (LoVo) cell line, with
approximately equal activity against the remaining three cell
lines.
The decomposition of O-benzyloxime indole (16d) was sus-
pected during cell culture experiments due to an observed
precipitation from the assay medium. As a control, two solutions of
16d in cell culture medium (10 and 100 mmol) were incubated at
37 ꢀC for seven days. Extraction with ethyl acetate and subsequent
NMR analysis showed approximately 50% breakdown of 16d to
oxime 16c. Other substituted oximes, including the cyclo-
hexylmethyl derivative 16e, were also subjected to this analysis and
no evidence for similar decomposition was observed.
The azepinone pharmacophore is present in several potent CDK
inhibitors including hymenialdisine (1, CDK2/cyclin A IC50 ¼ 70 nM)
[7] and kenpaullone (2, CDK2/cyclin A IC50 ¼ 0.68
mM) [12]. Six
compounds that showed favourable growth inhibition activity (9c,
9g, 9i, 16b, 16e and 16f) were assayed against cyclin dependent
kinase 2/cyclin A. Poor activity was observed with no compounds
inhibiting the enzyme by 50% or above at 10
mM (see Table 1).
5.2. Compound synthesis
Compound 9c was completely inactive, 9g, 9i, 16b and 16e
produced very weak activity but bromo-indole oxime 16f exhibited
5.2.1. 4,5-Dibromo-2-(trichloroacetyl)-1H-pyrrole (6a)
the best CDK2 inhibition (45% at 10
m
M). The binding mode of many
2-(Trichloroacetyl)-1H-pyrrole (6, 1.95 g, 9.18 mmol) was dis-
solved in THF (40 mL) and cooled to 0 ꢀC. Bromine (1 mL,
18.3 mmol) was added dropwise and stirred for 30 min. The reac-
tion was quenched with ice and extracted with ethyl acetate. The
combined extractions were washed with water, dried (MgSO4) and
concentrated under reduced pressure. Recrystallization (ethanol/
water) afforded the desired product as white crystals (3.16 g, 93%).
CDK inhibitors, including hymenialdisine, to their target enzyme
active site has been determined by x-ray crystallography [9].
Considering this, the N-methyl group of 9g, 9i and 16e might be
expected to block a hydrogen bond interaction and show reduced
CDK inhibition. Conversely, 9c, 16b and 16f, each containing an
additional hydrogen bond donor, would be predicted to show far
better (>>50%) CDK inhibition. This suggests that in contrast to
other azepinone derivatives, the substituted oximes derivatives
reported in this paper exert their anti-proliferative effects at
a primary target other than CDK2. A similar finding arose from
a study of the related tetrahydropyrrolo[3,2-c]azepin-4-one phar-
macophore [21].
mp ¼ 138e140 ꢀC; 1H NMR
d
7.42 (1H, s), 13.80 (1H, br s); 13C NMR
d
94.4, 101.2, 115.0, 122.8, 123.6, 171.3.
5.2.2. 3-(1H-pyrrole-2-carboxamido)propanoic acid (7)
2-(Trichloroacetyl)-1H-pyrrole (6, 675 mg, 3.18 mmol) was
dissolved in anhydrous THF (50 mL) under argon atmosphere

250
A.W. White et al. / European Journal of Medicinal Chemistry 56 (2012) 246e253
followed by anhydrous DIPA (2.50 mL, 13.0 mmol) and
b
-alanine
aqueous ethanol (1:1 EtOH/H2O, 40 mL) was heated at 60 ꢀC. Pyr-
roloazepine 8 (682 mg, 4.15 mmol) was added and the solution
heated at 60 ꢀC (2 h), and then heated at 100 ꢀC to evaporate the
alcohol. The precipitate was collected and dried under vacuum to
yield the desired oxime as an off-white solid (476 mg, 64%).
ethyl ester hydrochloride (980 mg, 6.38 mmol). The reaction was
refluxed (4 h) and then stirred at room temperature (3 h). The
solvents were removed under vacuum followed by addition of 1 M
HCl (50 mL). Solid residues were removed by filtration and the
aqueous solution was extracted with ethyl acetate, dried (MgSO4)
and concentrated under reduced pressure to afforded an oil which
was purified on a silica gel column (EtOAc/Petrol 60e80, gradient
from 10 to 100% EtOAc) to afford the ester as a white solid
(420.8 mg, 63%). mp ¼ 84e85 ꢀC; IR 3364, 3335, 2965, 1714, 1622,
mp ¼ 238e240 ꢀC; IR 3271, 3201, 2163, 1654, 1616 cmꢁ1
;
1H NMR
d
2.78 (2H, m), 3.20 (2H, m), 6.42 (1H, dd, J ¼ 2.5 2.4 Hz), 6.89 (1H,
dd, J ¼ 2.5, 2.4 Hz), 7.89 (1H, t, J ¼ 5.0 Hz), 10.09 (1H, s), 11.58 (1H, br
s); 13C NMR
d 29.5, 37.9, 106.5, 121.9, 122.5, 124.3, 152.4, 164.1; LRMS
(ESþ) m/z 202.0 [M þ Na]þ, 179.9 [M þ H]þ.
1528 cmꢁ1; 1H NMR
d
1.18 (3H, t, J ¼ 7.1 Hz), 2.54 (2H, t, J ¼ 7.0 Hz),
3.46 (2H, dt, J ¼ 7.0, 5.5 Hz), 4.07 (2H, q, J ¼ 7.1 Hz), 6.07 (1H, m),
5.2.7. (E)-4-(Benzyloxyimino)-4,5,6,7-tetrahydropyrrolo[2,3-c]
azepin-8(1H)-one (9b)
6.74 (1H, m), 6.84 (1H, m), 8.09 (1H, t, J ¼ 5.5 Hz), 11.45 (1H, s); 13C
NMR
d
14.4, 34.5, 35.2, 60.3, 108.9, 110.2, 121.6, 126.5, 161.1, 171.7;
Pyrroloazepine 8 (51 mg, 0.32 mmol), O-benzylhydroxylamine
(76 mg, 0.64 mmol) in anhydrous DMF (5 mL) with 1 drop of
glacial acetic acid were stirred at room temperature and moni-
tored by TLC until complete. The solvent was evaporated under
reduced pressure and the crude product purified by preparative
TLC (40% EtOAc/Petrol 60e80) affording the desired product as
a white solid (23 mg, 27%). mp ¼ 154e156 ꢀC; IR 3335, 3248,
LRMS (ESþ), m/z 211.1 [M þ H]þ, 233.1 [M þ Na]þ. The ester was
dissolved in THF (10 mL). After complete dissolution, lithium
hydroxide hydrate (252 mg, 6.00 mmol) was added to the stirred
solution. The mixture was refluxed and monitored by TLC until
complete. The resulting suspension was filtered, addition of water,
neutralisation with concentrated hydrochloric acid, extraction with
ethyl acetate, drying (MgSO4) and removal of the solvent afforded
the desired acid as a white solid (357 mg, 98%). mp ¼ 155e157 ꢀC;
2912, 2865, 1604 cmꢁ1
;
1H NMR (CDCl3)
d
3.05 (2H, m), 3.42 (2H,
m), 5.22 (2H, s), 6.58 (1H, t, J ¼ 5.1), 6.72 (1H, m), 6.95 (1H, m),
7.20e7.40 (5H, m), 10.09 (1H, br s); 13C NMR (CDCl3)
28.5, 37.8,
IR 3435, 3363, 3312, 2824, 1678, 1624 cmꢁ1; 1H NMR
d
2.49 (2H, t,
d
J ¼ 7.1 Hz), 3.40 (2H, dt, J ¼ 7.1 5.5 Hz), 6.07 (1H, m), 6.73 (1H, m),
75.4, 106.8, 121.4, 121.5, 122.6, 126.8, 127.2, 127.4, 136.8, 151.7,
6.84 (1H, m), 8.05 (1H, t, J ¼ 5.5 Hz), 11.43 (1H, s), 12.24 (1H, br s);
164.0; LRMS (ESþ) m/z 270.1 [M þ H]þ, 292.2 (100) [M þ Na]þ,
13C NMR
d
34.5, 35.2, 108.9, 110.2, 121.6, 126.5, 161.1, 173.3; LRMS
HRMS (ESþ) m/z 270.1234 ([M
þ
H]þ, 270.1237 calcd. for
(ESþ) m/z 204.9 [M þ Na]þ, (ESꢁ) m/z 181.0 [M ꢁ H]ꢁ.
C15H16N3O2).
5.2.3. 3-(4,5-Dibromo-1H-pyrrole-2-carboxamido)propanoicacid(7a)
Starting from 6a, compound 7a was obtained as a white solid
(68% over two steps) using a similar method to compound 7.
5.2.8. (E)-4-(Benzyloxyimino)-2,3-dibromo-4,5,6,7-tetrahydropyrrolo
[2,3-c]azepin-8(1H)-one (9c)
Starting from 8a, compound 9c was obtained as a white solid
mp ¼ 208e210 ꢀC; 1H NMR
d
2.49 (2H, t, J ¼ 7.1 Hz), 3.41 (2H, dt,
(18%) using a similar method to compound 9b. mp ¼ 216e218 ꢀC; IR
J ¼ 7.1, 5.5 Hz), 6.93(1H, d, J ¼ 2.7 Hz), 8.21 (1H, t, J ¼ 5.5 Hz), 12.28
3379, 3114, 2943, 1645, 1465 cmꢁ1; 1H NMR (CDCl3)
d 3.00 (2H, m),
(1H, br s), 12.70 (1H, br s); 13C NMR
d
34.2, 35.3, 98.1, 104.9, 113.0,
3.34 (2H, m), 5.19 (2H, s), 6.29 (1H, t, J ¼ 5.0 Hz), 7.22e7.40 (5H, m),
128.4, 159.3, 173.2; LRMS (ESꢁ) m/z 337.0, 338.9, 340.8 [M]ꢁ.
10.55 (1H, br s); 13C NMR (CDCl3)
d
31.3, 39.0, 77.2, 99.5, 109.8, 121.5,
125.3, 129.4, 129.8, 129.0, 138.1, 151.0, 164.1; LRMS (ESþ) m/z 425.8,
427.9, 429.8 [M þ H]þ, 447.8, 449.9, 451.9 [M þ Na]þ; Anal. found C
42.11%, H 3.22%, N 9.72%, (calcd. for C15H13Br2N3O2 C 42.18%, H
3.07%, N 9.83%).
5.2.4. 6,7-Dihydropyrrolo[2,3-c]azepine-4,8(1H,5H)-dione (8) [15]
In methanesulfonic acid (13 mL, 195.8 mmol) was dissolved
phosphorus pentoxide (2.02 g, 14.23 mmol) and heated to 80 ꢀC.
Once homogenous, carboxylic acid 7 (3.24 g, 17.78 mmol) was
added, the reaction stirred and monitored by TLC until complete.
The reaction was quenched with ice and exhaustively extracted
with ethyl acetate. The combined organic extractions were dried
(MgSO4) and concentration under vacuum to afford the desired
product as a white solid (1.58 g, 54%). mp ¼ 280e282 ꢀC; IR 3222,
5.2.9. 3-(1-Methyl-1H-pyrrole-2-carboxamido)propanoic acid (11)
To a solution of b-alanine ethyl ester HCl (3.49 g, 22.72 mmol)
and DMAP (4.26 g, 34.86 mmol) in anhydrous dichloromethane
(20 mL) at 0 ꢀC was added a solution of carboxylic acid 10 (2.58 g,
20.62 mmol) in anhydrous dichloromethane (15 mL) followed by
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide HCl (4.35 g,
22.69 mmol) dissolved in dichloromethane (10 mL). The solution
was stirred at 0 ꢀC (4 h), then at room temperature (20 h).
Concentration of the solvent under reduced pressure, addition of
water, extraction with dichloromethane, drying (MgSO4), and
concentration under reduced pressure afforded an oil which was
purified on a silica gel column (3% MeOH/dichloromethane) to
afford the ester as a white solid (4.02 g, 87%). mp ¼ 136e137 ꢀC; IR
2931, 1659, 1634 cmꢁ1
;
1H NMR
d
2.70 (2H, m), 3.35 (2H, m), 6.55
(1H, d, J ¼ 2.7 Hz), 6.99 (1H, d, J ¼ 2.7 Hz), 8.34 (1H, t, J ¼ 4.7 Hz),
12.18 (1H, br s); 13C NMR
d 36.9, 43.8, 109.9, 122.7, 123.9, 128.3,
162.6, 194.7; LRMS (ESþ) m/z 186.8 [M þ Na]þ; HRMS (ESþ)
165.0657 ([M þ H]þ, 165.0664 calcd. for C8H9N2O2).
5.2.5. 2,3-Dibromo-6,7-dihydropyrrolo[2,3-c]azepine-4,8(1H,5H)-
dione (8a) [22]
Starting from 7a, compound 8a was obtained as an off-white
solid (70%) using a similar method to compound 8. mp decom-
3348, 2983, 2944, 1730, 1639, 1546 cmꢁ1 1H NMR
; d 1.18 (3H, t,
J ¼ 7.1 Hz), 2.53 (2H, t, J ¼ 7.1 Hz), 3.40 (2H, dt, J ¼ 7.1, 5.5 Hz), 3.82
(3H, s), 4.06 (2H, q, J ¼ 7.1 Hz), 5.99 (1H, dd, J ¼ 3.9, 2.6 Hz), 6.72 (1H,
dd, J ¼ 3.9, 1.7 Hz), 6.88 (1H, dd, J ¼ 2.6, 1.7 Hz), 8.04 (1H, t,
posed >305 ꢀC; IR 3341, 3108, 2941,1657,1644 cmꢁ1; 1H NMR
d 2.77
(2H, m), 3.37 (2H, m), 8.55 (1H, t, J ¼ 5.0 Hz), 13.48 (1H, s); 13C NMR
d
36.4, 44.6, 99.9, 109.5, 120.8, 130.4, 161.4, 193.1; LRMS (CIþ) m/z
J ¼ 5.5 Hz); 13C NMR (CDCl3)
d 14.6, 34.7, 34.9, 37.2, 61.2,107.6,112.0,
321.0, 323.0, 325.0 [M þ H]þ, HRMS (EIþ) m/z 319.8792 ([M]þ,
126.0, 128.4, 162.2, 173.3; LRMS (ESþ) m/z 224.9 [M þ H]þ, 247.0
[M þ Na]þ. The ester was hydrolysed using the same method as
compound 7 to yield the product as an off-white solid (86%).
319.8791 calcd. for C8H6Br2N2O2).
5.2.6. (E)-4-(Hydroxyimino)-4,5,6,7-tetrahydropyrrolo[2,3-c]
azepin-8(1H)-one (9a)
A stirred solution of hydroxylamine hydrochloride (433 mg,
6.23 mmol) and sodium acetate trihydrate (1.13 g, 8.30 mmol) in
mp ¼ 136e137 ꢀC; IR 3398, 2976, 2546, 1721, 1714 cmꢁ1
;
1H NMR
d
2.47 (2H, t, J ¼ 7.1 Hz), 3.37 (2H, dt, J ¼ 7.1, 5.5 Hz), 3.82 (3H, s), 5.99
(1H, dd, J ¼ 3.9, 2.6 Hz), 6.73 (1H, dd, J ¼ 3.9, 1.7 Hz), 6.88 (1H, dd,
J ¼ 2.6, 1.7 Hz), 8.02 (1H, t, J ¼ 5.5 Hz), 12.2 (1H, br s); 13C NMR

A.W. White et al. / European Journal of Medicinal Chemistry 56 (2012) 246e253
251
d
34.4, 35.2, 36.5, 106.9, 112.5, 125.8, 128.0, 161.7, 173.3; LRMS (ESꢁ)
[M þ Na]þ; HRMS (ESþ) m/z 439.9589 ([M þ H]þ, 439.9604 calcd.
m/z 194.9 [M ꢁ H]ꢁ, 391 [2M]ꢁ.
for C16H16Br2N3O2).
5.2.10. 1-Methyl-6,7-dihydropyrrolo[2,3-c]azepine-4,8(1H,5H)-
dione (12) [23]
5.2.15. (E)-4-(Cyclohexylmethoxyimino)-1-methyl-4,5,6,7-
tetrahydropyrrolo[2,3-c]azepin-8(1H)-one (9h)
Starting from 11, compound 12 was obtained as a white solid
Starting from 9d, compound 9h was obtained as a pale yellow
solid (70%) using a similar method to compound 9f but using
bromomethylcyclohexane as the electrophile and 2% MeOH/
dicholoromethane as column eluent. mp ¼ 129e130 ꢀC; IR 3294,
(98%) using a similar method to compound 8. mp ¼ 194e196 ꢀC; IR
3339, 3103, 2902, 1669, 1629 cmꢁ1; 1H NMR (CDCl3)
d 2.88 (2H, m),
3.59 (2H, m), 4.02 (3H, s), 6.41 (1H, t, J ¼ 5.0 Hz), 6.77 (1H, d,
J ¼ 2.8 Hz), 6.83 (1H, d, J ¼ 2.8 Hz); 13C NMR (CDCl3)
d
38.06, 38.08,
3238, 2918, 1660, 1616 cmꢁ1; 1H NMR (CDCl3)
d 1.00e1.08 (2H, m),
44.4, 109.8, 126.3, 127.1, 129.2, 164.1, 195.2; LRMS (ESþ) m/z 201.0
[M þ Na]þ, 179.0 [M þ H]þ; Anal. found C 60.31%, H 5.71%, N
15.41%, (calcd. for C9H10N2O2.0$04H2O: C 60.42%, H 5.68%, N
15.66%).
1.20e1.33 (3H, m), 1.72e1.84 (6H, m), 2.97 (2H, m), 3.44 (2H, m),
3.95 (3H, s), 4.00 (2H, d, J ¼ 6.3 Hz), 6.15 (1H, t, J ¼ 6.0 Hz), 6.54 (1H,
d, J ¼ 2.7 Hz), 6.79 (1H, d, J ¼ 2.7 Hz); 13C NMR (CDCl3)
d 26.3, 27.0,
30.3, 32.3, 37.3, 38.0, 38.9, 80.3, 106.6, 123.3, 124.3, 128.8, 153.2,
165.4; LRMS (CIþ) m/z 290.2 [M þ H]þ; HRMS (ESþ) m/z 290.1859
([M þ H]þ, 290.1863 calcd. for C16H24N3O2); Anal. found C 65.38%, H
8.03%, N 14.22%, (calcd. for C16H23N3O2.0$2H2O C 65.59%, H 8.05%, N
14.34%).
5.2.11. (E)-4-(Hydroxyimino)-1-methyl-4,5,6,7-tetrahydropyrrolo
[2,3-c]azepin-8(1H)-one (9d)
Starting from 12, compound 9d was obtained as an off-white
solid (82%) using a similar method to compound 9a. mp ¼ 200e
202 ꢀC; IR 3473, 3266, 2907, 1657, 1651 cmꢁ1
;
1H NMR
d
2.68 (2H,
5.2.16. (E)-2,3-dibromo-4-(cyclohexylmethoxyimino)-1-methyl-
4,5,6,7-tetrahydropyrrolo[2,3-c]azepin-8(1H)-one (9i)
m), 3.16 (2H, m), 3.75 (3H, s), 6.25 (1H, d, J ¼ 2.6 Hz), 6.92 (1H, d,
J ¼ 2.6 Hz), 7.91 (1H, t, J ¼ 5.6 Hz),10.91 (1H, s); 13C NMR
d
31.6, 36.5,
Starting from 9e, compound 9i was obtained as an off-white
solid (20%) using a similar method to compound 9f but using
iodomethylcyclohexane as the electrophile and preparative TLC (2%
MeOH/dichloromethane) to purify the title compound. mp ¼ 133e
37.6, 105.4, 123.3, 123.6, 128.3, 152.8, 164.2; LRMS (ESþ) m/z 194.1
[M þ H]þ, 216.0 [M þ Na]þ, 408.9 [2M þ Na]þ.
5.2.12. (E)-2,3-dibromo-4-(hydroxyimino)-1-methyl-4,5,6,7-
tetrahydropyrrolo[2,3-c]azepin-8(1H)-one (9e)
135 ꢀC; IR 3272, 3067, 2920, 2853, 1660 cmꢁ1 1H NMR (CDCl3)
;
d
1.00e1.08 (2H, m), 1.25e1.29 (3H, m), 1.74e1.85 (6H, m), 2.99 (2H,
Starting from 9d, compound 9e was obtained as an off-white
solid (75%) using a similar method to compound 6a.ꢁm1 p decom-
m), 3.43 (2H. m), 3.97 (3H, s), 4.05 (2H, d, J ¼ 6.5 Hz), 6.46 (1H, t,
J ¼ 6.0 Hz); 13C NMR (CDCl3)
d 26.3, 27.1, 30.2, 33.7, 36.3, 38.0, 38.7,
posed >300 ꢀC; IR 3306, 3203, 2984, 1651, 1640 cm
;
1H NMR
80.6, 98.9, 114.9, 122.1, 125.2, 150.2, 164.4; LRMS (ESþ) m/z 446.0,
447.8, 450.0 [M þ H]þ; 467.9, 469.9, 472.1 [M þ Na]þ; HRMS (ESþ)
m/z 446.0075 ([M þ H]þ, 446.0079 calcd. for C16H22Br2N3O2).
d
2.78 (2H, m), 3.22 (2H, m), 3.81 (3H, s), 8.25 (1H, t, J ¼ 5.9 Hz),
11.45 (1H, s); 13C NMR
d 32.4, 34.8, 36.4, 96.4, 112.2, 120.0, 125.4,
149.6, 162.2; LRMS (ESꢁ) m/z 347.7, 349.9, 351.8 [M ꢁ H]ꢁ.
5.2.17. 3-(1H-indole-2-carboxamido)propanoic acid (14)
5.2.13. (E)-4-(Benzyloxyimino)-1-methyl-4,5,6,7-tetrahydropyrrolo
[2,3-c]azepin-8(1H)-one (9f)
Starting from 13, compound 14 was obtained as a pale yellow
solid (82% over two steps) using a similar method to compound 11
To a solution of 9d (94 mg, 0.49 mmol) and potassium tert-
butoxide (55 mg, 0.49 mmol) in anhydrous THF (10 mL) under
argon was added benzyl bromide (0.06 mL, 0.54 mmol). The
mixture was stirred at room temperature and monitored by TLC
until complete. The solution was concentrated and dichloro-
methane (20 mL) was added. The organic layer was washed with
water, dried (MgSO4), and evaporated under reduced pressure to
afford the crude product which was purified on a silica gel column
(5% MeOH/dichloromethane). The desired product was isolated as
a white solid (83 mg, 60%). mp ¼ 143e144 ꢀC; IR 3286, 3188, 3064,
mp 194e198 ꢀC; 1H NMR
d 2.54e2.59 (2H, m), 3.48e3.54 (2H, m),
7.05 (1H, dd, J ¼ 7.2, 7.8 Hz), 7.12 (1H, s), 7.20 (1H, dd, J ¼ 7.2 Hz),
7.45 (1H, d, J ¼ 7.8 Hz), 7.63 (1H, d, J ¼ 7.3 Hz), 8.56 (1H, t, J ¼ 5.1 Hz),
11.58 (1H, s), 12.38 (1H, br s); 13C NMR
d 34.3, 65.3, 102.8, 112.6,
120.0, 121.8, 123.6, 127.4, 132.0, 136.8, 161.5, 173.2; LRMS (ESꢁ) m/z
231.0 [M ꢁ H]ꢁ.
5.2.18. 3,4-Dihydroazepino[3,4-b]indole-1,5(2H,10H)-dione (15) [17]
Starting from 14, compound 15 was obtained as a yellow solid
(58%) using a similar method to compound 8 with purification by
recrystallisation from acetone and petrol 60e80. mp 282e284 ꢀC;
2933, 1658 cmꢁ1; 1H NMR (CDCl3)
d 3.01 (2H, m), 3.43 (2H, m), 3.96
(3H, s), 5.25 (2H, s), 6.07 (1H, t, J ¼ 5.9 Hz), 6.55 (1H, d, J ¼ 2.7 Hz),
1H NMR
d 2.84e2.88 (2H, m), 3.46e3.51 (2H, m), 7.25e7.38 (2H,
6.79 (1H, d, J ¼ 2.7 Hz), 7.30-7.50 (5H, m, Ph); 13C NMR (CDCl3)
m), 7.55 (1H, d, J ¼ 8.1 Hz), 8.33 (1H, d, J ¼ 7.8 Hz), 8.79 (1H, t,
d
32.4, 37.4, 38.8, 76.2, 106.7, 123.5, 123.9, 128.3, 128.6, 128.8, 138.3,
J ¼ 5.0), 12.50 (1H, s); 13C NMR
d 34.3, 35.6, 102.8, 112.6, 120.0, 121.8,
123.6, 127.4, 132.0, 136.7, 161.5, 173.2; HRMS (ESþ) m/z 215.0813
154.1, 160.2, 165.4; LRMS (CIþ) m/z 284.2 [M þ H]þ, 178.1
[MꢁOBn]þ; HRMS (ESþ) m/z 284.1394 ([M þ H]þ, 284.1394 calcd.
for C16H18N3O2); Anal. found C 67.72%, H 6.23%, N 14.64%, (calcd. for
C16H17N3O2 C 67.83%, H 6.05%, N 14.83%).
([M þ H]þ, 215.0815 calcd. for C12H11N2O2).
5.2.19. (E)-5-(Hydroxyimino)-2,3,4,5-tetrahydroazepino[3,4-b]
indol-1(10H)-one (16a)
5.2.14. (E)-4-(Benzyloxyimino)-2,3-dibromo-1-methyl-4,5,6,7-
tetrahydropyrrolo[2,3-c]azepin-8(1H)-one (9g)
Indoloazepinone 15 (1.00 g, 4.67 mmol) and hydroxylamine
hydrochloride (3.24 g, 46.68) were dissolved in THF (75 mL) under
nitrogen at ꢁ10 ꢀC. Boron trifluoride diethyletherate (4.00 mL,
31.6 mmol) was added, followed by pyridine (6.00 mL, 74.2 mmol).
The mixture was stirred at ꢁ10 ꢀC for 1 h, then allowed to warm to
room temperature and left stirring for 2 days. The solvents were
evaporated, and the mixture dissolved in methanol. Water was
added and the solution was left overnight in a beaker. The product
was recovered as a yellow precipitate (0.46 g, 43%). mp 258e
Starting from 9e, compound 9g was obtained as an off-white
solid (20%) using a similar method to compound 9f but using
preparative TLC (5% MeOH/dichloromethane) to purify the title
compound. mp
¼
184e186 ꢀC; IR 3282 3062, 2919, 2854,
1660 cmꢁ1; 1H NMR (CDCl3)
d 2.95 (2H, m), 3.34 (2H, m), 3.88 (3H,
s), 5.20 (2H, s), 6.37 (1H, t, J ¼ 6.0 Hz), 7.20e7.40 (5H, m); 13C NMR
(CDCl3)
d 33.8, 36.4, 38.6, 77.2, 99.0, 115.1, 121.9, 125.3, 128.3, 128.8,
128.9, 138.2, 151.1, 164.4; LRMS (ESþ) m/z 461.8, 463.9, 465.8
260 ꢀC; 1H NMR
d 2.85e2.89 (2H, m), 3.47e3.52 (2H, m), 7.26e

252
A.W. White et al. / European Journal of Medicinal Chemistry 56 (2012) 246e253
7.38 (2H, m), 7.56 (1H, d, J ¼ 8.0 Hz), 8.33 (1H, d, J ¼ 7.9 Hz), 8.79
1644, 2922, 3353 cmꢁ1 1H NMR
; d 2.88e2.91 (2H, m), 3.27e3.32
(1H, t, J ¼ 5.0), 11.12 (1H, s), 11.80 (1H, s); 13C NMR
d
30.5, 37.8, 112.5,
(2H, m), 3.93 (3H, s), 7.18 (1H, dd, J ¼ 8.0 6.9 Hz), 7.35 (1H, dd,
112.7,120.9,124.5,124.7,125.1,130.0,136.6,154.0,164.6; LRMS (EIþ)
J ¼ 8.3 6.9 Hz), 7.57 (1H, d, J ¼ 8.3 Hz), 8.09 (1H, d, J ¼ 8.0 Hz), 8.44
m/z ¼ 229.1 [M]þ.
(1H, t, J ¼ 5.9 Hz), 11.23 (1H, s); 13C NMR
d 32.8, 37.0, 45.3, 111.4,
115.1, 123.1, 125.4, 135.1, 138.4, 159.9, 162.8, 196.1; HRMS (EIþ) m/z
243.1007 ([M]þ, 243.1008 calcd. for C13H13N3O2); Anal. found C
63.33%, H 5.33%, N 16.83%, (calcd. for C13H13N3O2.0.13H2O C 63.60%,
H 5.44%, N 17.12%).
5.2.20. (E)-5-(Benzyloxyimino)-2,3,4,5-tetrahydroazepino[3,4-b]
indol-1(10H)-one (16b)
Starting from 15, compound 16b was obtained as a creamy white
solid (27%) using a similar method to compound 16a with O-ben-
zylhydroxylamine as the nucleophile. mp 204 ꢀC; IR 1643, 3271,
5.2.24. (E)-5-(Benzyloxyimino)-10-methyl-2,3,4,5-tetrahydroazepino
[3,4-b]indol-1(10H)-one (16d)
3373 cmꢁ1 1H NMR
; d 3.01e3.04 (2H, m), 3.29e3.33 (2H, m), 5.26
(2H, s), 7.10 (1H, dd, J ¼ 8.17.2 Hz), 7.26 (1H, dd, J ¼ 7.8 7.2 Hz), 7.32e
Starting from 16c, compound 16d was obtained as a light brown
solid (15%) using a similar method to compound 9f with purifica-
tion by column chromatography (1:1 petrol 60e80/ethyl acetate).
7.48 (6H, m), 8.12 (1H, d, J ¼ 8.1 Hz), 8.40 (1H, t, J ¼ 5.1 Hz), 11.93
(1H, s); 13C NMR
d 31.1, 37.6, 75.8, 111.5, 112.6, 121.3, 124.4, 124.8,
124.9, 128.0, 128.5, 128.7, 130.9, 136.5, 138.6, 155.1, 164.3; HRMS
mp 156e158 ꢀC; 1H NMR
d 2.91e2.96 (2H, m), 3.28e3.37 (2H, m),
(EIþ) m/z 320.1394 ([M þ H]þ, 320.1394 calcd. for C19H18N3O2);
4.01 (3H, s), 5.18 (2H, s), 7.17 (1H, dd, J ¼ 7.8 Hz), 7.31e7.53 (6H, m),
Anal. found
C
70.90%,
H
5.45%,
N
12.81% (calcd. for
7.58 (1H, d, J ¼ 7.8 Hz), 8.00 (1H, d, J ¼ 7.2 Hz), 8.64 (1H, t,
C19H17N3O2.0.08EtOAc C 71.11%, H 5.44%, N 12.88%).
J ¼ 5.1 Hz); 13C NMR
d 32.1, 33.4, 37.3, 75.8, 110.8, 112.2, 121.6, 123.6,
123.8,125.0,128.1,128.4,128.7,131.1,138.6,138.7,154.5,164.1; LRMS
5.2.21. 3-(1-Methyl-1H-indole-2-carboxamido)propanoic acid (18)
[17]
(ESþ) m/z 356.1 [M þ Na]þ.
Carboxylic acid (17) (2.61 g, 14.90 mmol), was dissolved in THF
(150 mL) under nitrogen and triethylamine (6.30 mL, 77.4 mmol)
was added. The mixture was cooled to 0 ꢀC before adding oxalyl
chloride (2.90 mL, 33.2 mmol) and one drop of DMF. After 5 min,
the mixture was allowed to return to room temperature and was
stirred under nitrogen (24 h). The solvents were evaporated and the
mixture suspended in THF (50 mL). This was added to a cooled
5.2.25. (E)-5-(Cyclohexylmethoxyimino)-10-methyl-2,3,4,5-
tetrahydroazepino[3,4-b]indol-1(10H)-one (16e)
Starting from 16c, compound 16e was obtained as a pale brown
solid (16%) using a similar method to compound 9f but using bro-
momethylcyclohexane as the electrophile. mp 154e156 ꢀC; 1H NMR
d
0.95e1.31 (6H, m), 1.70e1.80 (5H, m), 2.89e2.92 (2H, m), 3.26e
3.32 (2H, m), 3.94 (3H, s), 4.01 (2H, d, J ¼ 6.2 Hz), 7.21 (1H, dd,
(0 ꢀC) solution of
b-alanine ethyl ester hydrochloride (11.40 g;
J ¼ 8.1 6.8 Hz), 7.37 (1H, dd, J ¼ 8.4 6.8 Hz), 7.59 (1H, d, J ¼ 8.4 Hz),
74.21 mmol) and triethylamine (59.0 mL; 724.6 mmol) in THF
(400 mL). The mixture was stirred at room temperature (24 h). The
solvents evaporated, saturated sodium bicarbonate solution was
added and extracted with ethyl acetate. The combined extracts
were washed with 1 M HCl (50 mL) and dried (MgSO4). The solvent
was evaporated to recover the ester as a dark oil (3.28 g, 80%). 1H
8.12 (1H, d, J ¼ 8.1 Hz), 8.48 (1H, t, J ¼ 6.0 Hz); 13C NMR
d 25.7, 26.5,
29.7, 32.1, 33.3, 37.7, 79.4, 110.8, 112.4, 121.6, 123.5, 123.8, 125.0,
131.0, 138.6, 153.7, 164.0; MS (ESþ) m/z 340.2023 ([M þ H]þ,
340.2020 calcd. for C20H26N3O2); Anal. found C 68.87%, H 7.48%, N
11.06%, (calcd. for C20H25N3O2.0.5EtOAc C 68.90%, H 7.62%, N
10.96%).
NMR
d
1.22 (3H, t, J ¼ 7.1 Hz), 2.63 (2H, t, J ¼ 7.0 Hz), 3.53 (2H, q,
J ¼ 7.0 Hz), 4.01 (3H, s), 4.11 (2H, q, J ¼ 7.1 Hz), 7.08 (1H, s), 7.13 (1H,
5.2.26. 7-Bromo-3,4-dihydroazepino[3,4-b]indole-1,5(2H,10H)-dione
(20)
t, J ¼ 7.1 Hz), 7.30 (1H, dd, J ¼ 7.1, 8.3 Hz), 7.54 (1H, d, J ¼ 8.4 Hz), 7.66
(1H, d, J ¼ 7.9 Hz), 8.61 (1H, t, J ¼ 5.4 Hz); 13C NMR
d
13.2, 29.3, 33.0,
Indole 15 (0.20 g, 0.93 mmol) was dissolved in DMF (8 mL).
N-Bromosuccinamide (0.18 g, 0.93 mmol) dissolved in DMF (2 mL)
was added and the reaction was stirred overnight. Ice was added,
the resulting precipitate filtered and washed with water to recover
the product as a yellow solid (0.24 g, 88%). mp > 360 ꢀC; 1H NMR
59.9, 102.8, 109.1, 119.4, 120.8, 123.0, 125.0, 130.8, 138.0, 161.5, 171.8;
LRMS (ESþ) m/z 275.0 [M þ H]þ. The ester was hydrolysed using the
same method as compound 7 to yield the product as a brown solid
(97%). mp 144e148 ꢀC; 1H NMR
d 2.51e2.57 (2H, m), 3.44e3.50 (2H,
m), 3.99 (3H, s), 7.06 (1H, s), 7.10 (1H, dd, J ¼ 7.2, 7.7 Hz), 7.28 (1H,
d 2.84e2.87 (2H, m), 3.43e3.48 (2H, m), 7.46e7.53 (2H, m), 8.45
dd, J ¼ 7.3, 7.9 Hz), 7.52 (1H, d, J ¼ 8.3 Hz), 7.63 (1H, d, J ¼ 7.8 Hz),
(1H, s), 8.85 (1H, t, J ¼ 5.2 Hz), 12.69 (1H, s); 13C NMR
d 36.9, 44.3,
8.56 (1H, t, J ¼ 5.3 Hz), 12.30 (1H, br s); 13C NMR
d
31.7, 34.1, 35.5,
113.5, 15.2, 115.8, 125.2, 128.0, 128.1, 134.7, 136.0, 162.4, 195.6; HRMS
104.6, 110.8, 120.4, 121.9, 123.8, 125.9, 162.2, 173.2; LRMS (ESꢁ) m/z
(EIþ) m/z 291.9842 ([M]þ, 291.9847 calcd. for C12H9BrN2O2).
244.7 [M ꢁ H]ꢁ.
5.2.27. (E)-7-bromo-5-(hydroxyimino)-2,3,4,5-tetrahydroazepino
[3,4-b]indol-1(10H)-one (16f)
5.2.22. 10-Methyl-3,4-dihydroazepino[3,4-b]indole-1,5(2H,10H)-
dione (19) [17]
Starting from 20, compound 16f was obtained as an off-white
solid (57%) using a similar metꢁh1od to compound 16a. mp 274e
Starting from 18, compound 19 was obtained as a brown solid
(82%) using a similar method to compound 8 with purification by
column chromatography (5% EtOAc/Petrol 60e80). mp 207e
276 ꢀC; IR 1633, 3291, 3404 cm
; d 2.95e2.98 (2H, m),
1H NMR
3.30e3.45 (2H, m), 7.40e7.41 (2H, m), 8.42e8.44 (2H, m), 11.25 (1H,
209 ꢀC; 1H NMR
d
2.79e2.82 (2H, m), 3.40e3.46 (2H, m), 4.03
s), 12.03 (1H, br s); 13C NMR
d
30.0, 37.7, 112.1, 113.4, 114.6, 126.6,
(3H, s), 7.32 (1H, dd, J ¼ 7.1 Hz), 7.43 (1H, dd, J ¼ 7.1, 8.1 Hz), 7.68
127.3, 131.2, 135.3, 153.7, 164.1; HRMS (ESþ) m/z 308.0029
(1H, d, J ¼ 8.0 Hz), 8.30 (1H, d, J ¼ 8.0), 8.84 (1H, t, J ¼ 5.2 Hz); 13C
([M þ H]þ, 308.0030 calcd. for C12H11BrN3O2).
NMR (CDCl3) d 33.2, 38.2, 45.5, 110.7, 124.3, 125.7, 126.3, 133.5, 139.1,
159.9, 164.4, 195.9; HRMS (ESþ) m/z 229.0978 ([M þ H]þ, 229.0977
5.3. Growth inhibition assay
calcd. for C13H13N2O2).
The four human cancer cell lines used; A549 (lung), LoVo
(colorectal) MCF-7 (breast) and PC3 (prostate); were routinely
passaged and grown in a humid atmosphere with 5% CO2, at 37 ꢀC.
All cancer cell lines were cultured in Dulbecco’s Modified Eagle
Medium containing 10% heat inactivated foetal calf serum (A549
5.2.23. (E)-5-(Hydroxyimino)-10-methyl-2,3,4,5-tetrahydroazepino
[3,4-b]indol-1(10H)-one (16c)
Starting from 19, compound 16c was obtained as a white solid
(82%) using a similar method to compound 9a. mp 202e204 ꢀC; IR

A.W. White et al. / European Journal of Medicinal Chemistry 56 (2012) 246e253
253
and MCF7) or 10% foetal calf serum (LoVo and PC3). Exponentially
Acknowledgements
growing cells were used in all experiments. Cell viability was
measured using a colormetric MTT assay. Cells (3e10 ꢂ 103/well)
were seeded into 96-well plates and incubated at 37 ꢀC for 24 h.
The authors gratefully acknowledge the Welsh Office for
Research and Development (WORD) for partial financial support,
the EPRSC National Mass Spectrometry Service Centre, University
of Swansea, UK, for high resolution mass spectrometry and Huw
Mottram for conducting the proliferation assays.
Media was removed and replaced with 150
mL of media containing
test compounds at concentrations of 0.1, 1, 10, 50 and 100
mM
(diluted from 10 mM DMSO stock solution) and incubated for
a further 96 h. After this time, media was removed and replaced
with MTT solution in phenol red free RPMI (0.5 mg/mL). Absor-
bance was measured at 540 nm using a microplate reader spec-
trophotometer, inhibition of proliferation was assessed as the
percentage reduction of absorbance of treated cells versus media-
only controls and are the mean of at least three experiments. The
concentration of test compound that reduced the growth of cells by
50% (GI50) was calculated using a non-linear regression analysis in
Prism (Graphpad software).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.08.022.
References
[1] P. Russo, C. Nastrucci, A. Cesario, Curr. Med. Chem. 18 (2011) 3551e3562.
[2] R. Montaser, H. Luesch, Future Med. Chem. 3 (2011) 1475e1489.
[3] S. Jain, L.T. Vahdat, Clin. Cancer Res. 17 (2011) 6615e6622.
[4] E. Hamel, Pharmacol. Ther. 55 (1992) 31e51.
5.4. CDK2/cyclin A assay
[5] M. D’Incalci, C.M. Galmarini, Mol. Cancer Ther. 9 (2010) 2157e2163.
[6] B. Forte, B. Malgesini, C. Piutti, F. Quartieri, A. Scolaro, G. Papeo, Mar. Drugs 7
(2009) 705e753.
[7] G. Cimino, S. De Rosa, S. De Stefano, L. Mazzarella, R. Puliti, G. Sodano,
Tetrahedron Lett. 23 (1982) 767e768.
[8] T.N.T. Nguyen, J.J. Tepe, Curr. Med. Chem. 16 (2009) 3122e3143.
[9] L. Meijer, A.M.W.H. Thunnissen, A.W. White, M. Garnier, M. Nikolic, L.-H. Tsai,
J. Walter, K.E. Cleverley, P.C. Salinas, Y.-Z. Wu, J. Biernat, E.-M. Mandelkow, S.-
H. Kim, G.R. Pettit, Chem. Biol. 7 (2000) 51e63.
[10] C. Lange, E. Mix, J. Frahm, A. Glass, J. Mueller, O. Schmitt, A.-C. Schmoele,
K. Klemm, S. Ortinau, R. Huebner, M.J. Frech, A. Wree, A. Rolfs, Neurosci. Lett.
488 (2011) 36e40.
[11] V. Sharma, J.J. Tepe, Bioorg. Med. Chem. Lett. 14 (2004) 4319e4321.
[12] D.W. Zaharevitz, R. Gussio, M. Leost, A.M. Senderowicz, T. Lahusen, C. Kunick,
L. Meijer, E. Sausville, Cancer Res. 59 (1999) 2566e2569.
[13] C. Schultz, A. Link, M. Leost, D.W. Zaharevitz, R. Gussio, E.A. Sausville, L. Meijer,
C. Kunick, J. Med. Chem. 42 (1999) 2909e2919.
[14] H. Annoura, T. Tatsuoka, Tetrahedron Lett. 36 (1995) 413e416.
[15] G. Papeo, H. Posteri, D. Borghi, M. Varasi, Org. Lett. 7 (2005) 5641e5644.
[16] K. Wieking, M. Knockaert, M. Leost, D.W. Zaharevitz, L. Meijer, C. Kunick, Arch.
Pharm. 335 (2002) 311e317.
A CDK2/cyclin A assay kit, containing recombinant protein
expressed in Sf21 cells, was purchased from Upstate Inc., Lake
Placid, NY, USA. Radiolabelled ATP was purchased from Amer-
sham, UK. The enzyme was diluted in assay kit buffer (10% v/v)
and 32P-ATP was diluted in kit buffer (1% v/v) containing magne-
sium chloride (750
m
M) and cold ATP (5
mM). A range of duplicate
dilutions of the test inhibitor (100
mMe10 nM) were made from
a 10 mM DMSO stock solution using sterile water with 0.1% DMSO
as diluent. Assays were performed in 1.5 mL Eppendorf tubes as
follows: add 5
diluted histone-H1 on ice. CDK2/cyclin A solution (2.5
was added and removed from ice, followed by addition of the
inhibitor solutions. Finally, 10
L of prediluted 32P-ATP was added
to each assay and incubated for 10 min at 30 ꢀC with shaking.
Assay solution (20 L) was spotted onto numbered phospho-
mL of kit buffer per assay on ice, followed by 5
mL of
mL of 50 ng)
m
m
cellulose paper (from kit) and allowed to air dry. The filters were
transferred to 50 mL Falcon tubes containing 0.75% phosphoric
acid and washed on a rotating mixer for 5 min. The radioactive
solution was disposed and washes repeated twice. The filters were
finally washed with acetone for 5 min. Air dried filters were
transferred to scintillation tubes for counting in 5 mL scintillation
cocktail. Percentage inhibition values were calculated by compar-
ison to an untreated control and are the mean of duplicate
experiments.
[17] V. Sharma, T.A. Lansdell, G. Jin, J.J. Tepe, J. Med. Chem. 47 (2004) 3700e3703.
[18] Y. Wan, W. Hur, C.Y. Cho, Y. Liu, F.J. Adrian, O. Lozach, S. Bach, T. Mayer,
D. Fabbro, L. Meijer, N.S. Gray, Chem. Biol. 11 (2004) 247e259.
[19] B. Portevin, R.M. Golsteyn, A. Pierre, G. De Nanteuila, Tetrahedron Lett. 44
(2003) 9263e9265.
[20] H. Cho, S. Matsuki, Heterocycles 43 (1996) 127e131.
[21] R. Martinez, J.G. Avila, M.T. Ramirez, A. Perez, A. Martinez, Bioorg. Med. Chem.
14 (2006) 4007e4016.
[22] Y. Wan, N.S. Gray, Int. Pat. Appl. (2004). WO2004098520.
[23] X.-C. Zeng, S.-H. Xu, J. Gu, D.-S. Deng, Acta Crystallogr.
E 61 (2005)
O1805eO1806.