Synthesis and in vitro binding of N,N-dialkyl-2-phenylindol-3-yl-glyoxylamides for the peripheral benzodiazepine binding sites

Article abstract of DOI:10.1016/j.bmc.2006.01.039

A series of N,N-dialkyl-2-phenylindol-3-ylglyoxylamides bearing the halogens iodine and bromine were synthesised and their binding affinity for the peripheral benzodiazepine binding sites (PBBS) in rat kidney mitochondrial membranes was evaluated using [<

Full text of DOI:10.1016/j.bmc.2006.01.039

Bioorganic & Medicinal Chemistry 14 (2006) 3938–3946
Synthesis and in vitro binding of N,N-dialkyl-2-phenylindol-3-
yl-glyoxylamides for the peripheral benzodiazepine binding sites
Taryn P. Homes,a Filomena Mattner,b Paul A. Kellera and Andrew Katsifisb,*
aDepartment of Chemistry, University of Wollongong, NSW 2500, Wollongong, Australia
bRadiopharmaceutical Research Institute, Australian Nuclear Science and Technology Organisation,
Menai NSW 2234, Sydney, Australia
Received 9 November 2005; revised 16 January 2006; accepted 20 January 2006
Available online 15 February 2006
Abstract—A series of N,N-dialkyl-2-phenylindol-3-ylglyoxylamides bearing the halogens iodine and bromine were synthesised and
their binding affinity for the peripheral benzodiazepine binding sites (PBBS) in rat kidney mitochondrial membranes was evaluated
using [3H]PK11195. Central benzodiazepine receptor (CBR) affinities were also evaluated in rat cortices using [3H]flumazenil to
determine their selectivity for PBBS over CBR. The tested compounds had PBBS binding affinities (IC50) ranging from 7.86 to
618 nM, with all compounds showing high selectivity over the CBR (CBR IC50 > 5000 nM). Among the 12 compounds tested, those
with a diethylamide group were the most potent. The highest affinity iodinated PBBS ligand, N,N-diethyl-[5-chloro-2-(4-iodophe-
nyl)indol-3-yl]glyoxylamide (4c), was radiolabelled with iodine-123. This high affinity and selective radioligand may be useful for
imaging neurodegeneration, inflammation and tumours using single photon emission computed tomography.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
The development of specific radiotracers for the PBBS
has not only contributed to the elucidation of the recep-
tor’s biochemical functions, but when imaged with posi-
tron emission tomography (PET) and single photon
emission computed tomography (SPECT) provides a
means by which these changes can be monitored and
correlated to disease.10–13 A diverse range of chemical
structures have been shown to bind to various compo-
nents of the PBBS. The isoquinoline carboxamide ligand
PK 11195, the classical 1,4-benzodiazepine, Ro 5-4864
and the imidazopyridine Alpidem (Fig. 1) have been
shown to bind to various binding domains of the
PBR. [11C]PK 11195 has been used to study the PBBS
in vivo using PET to detect brain inflammation in hu-
mans and to image early Alzheimer’s disease.14
The peripheral benzodiazepine binding sites (PBBS)
(also termed the peripheral benzodiazepine receptors
or PBR) are multimeric protein complexes localised
mainly on the outer mitochondrial membranes of cells
in peripheral organs such as the kidney, heart and
steroid producing cells of the adrenals, testes and ova-
ries.1,2 The PBBS are pharmacologically and anatomi-
cally different to the central benzodiazepine receptors
(CBR). Within the normal central nervous system,
PBBS are expressed at low levels mainly on the choroid
plexus, ependymal lining of the ventricles and the olfac-
tory bulb.3 Dramatic upregulation of PBBS has been
reported in such diverse neuropathologic states includ-
ing Huntington’s and Alzheimer’s diseases and multiple
sclerosis.4,5 Significantly enhanced PBBS expression has
also been observed in melanoma, breast, prostate, ovar-
ian and glial tumours.6–9
More recently, imidazopyridines15 and pyridazines16
and indoles such FGIN-1-2717,18 (Fig. 1) have all been
reported to bind to the PBBS. Several of these ligands
have been radiolabelled with iodine-123, for potential
imaging using SPECT.16,19,20
N,N-Dialkyl-2-phenylindolyl-3-glyoxylamides (Fig. 2)
represent a new class of potent PBBS ligands, whose
development was based on conformationally con-
strained analogues of 2-phenylindole-3-acetamides.21
These compounds offer the potential for radiolabelling
Keywords: Peripheral benzodiazepine binding sites; N,N-Dialkyl-2-phen-
ylindol-3-ylglyoxylamides; SPECT; Radioiodination
*
.
Corresponding author. Tel.: +612 9717 9094; fax: +612 9717 9262;
e-mail: akx@ansto.gov.au
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.01.039

T. P. Homes et al. / Bioorg. Med. Chem. 14 (2006) 3938–3946
3939
R3
CH
N
3 O
CH3
O
H3C
H3C
O
R4
i
CH3
R3
N
N
N
Cl
R4 = H or Cl
N
H
N
CH3
Cl
R3 = Br or I
1a, R3 = I, R4 = Cl
1b, R3 = I, R4 = H
1c, R3 = Br, R4 = Cl
Cl
iii
Ro 5-4864
PK 11195
ii
CH3
O
Cl
R4
R4
CH3
O
H3C
iv
N
H
N
H
CH3
R3
N
O
N
O
R3
3a-d
2a-d
For 2 and 3
Cl
F
N
N
v
a, R3 = I, R4 = Cl
b, R3 = I, R4 = H
c, R3 = Br, R4 = Cl
d, R3 = Br, R4 = H
Cl
N
H
R1
O
N
O
R2
Alpidem
FGIN-1-27
R4
Figure 1. Structures of PBBS ligands.
R1 and R2 = hexyl,
propyl, ethyl, or
methyl
N
H
R3
R1
4a-l
N R2
O
Scheme 1. Reagents: (i) Ph-NHNH2 or 4-Cl-PhNHNH2ÆHCl, ethanol,
CH3CO2H; (ii) to synthesis 2a–c, polyphosphoric acid; (iii) to
synthesise 2d, Ph-NHNH2, polyphosphoric acid; (iv) ClCOCOCl,
anhydrous ether; (v) NH(alkyl)2, triethylamine, anhyd toluene.
O
R4
R3
N
H
N,N-dialkyl-2-phenylindol-3-ylglyoxylamides
O
O
N
N
Cl
Cl
Figure 2. Structure of N,N-dialkyl-2-phenylindol-3-ylglyoxylamides.
O
O
i
N
H
N
H
Sn(CH3)3
Br
with radionuclides for both PET and SPECT imaging.
In this investigation, we synthesised and tested several
of halogenated derivatives of these systems with the
aim of radiolabelling with iodine-123 for studying PBBS
using SPECT.
4k
5
ii
O
N
Cl
2. Chemistry
O
N
H
The N,N-dialkyl-2-phenylindol-3-ylglyoxylamides 4a–l
were synthesised following literature methods21 for simi-
lar compounds and is outlined in Scheme 1. Briefly, 2-(4-
bromophenyl)indole 2d was synthesised in one step,
from 4-bromoacetophenone and phenylhydrazine with
polyphosphoric acid,22 while indoles 2a–c were synthe-
sised in two steps via their phenylhydrazone derivatives
1a–c.23 The phenylhydrazones were formed by reacting
4-bromo or 4-iodo acetophenone with phenylhydrazine
or 4-chlorophenylhydrazine hydrochloride, followed by
cyclisation by the Fischer indole synthesis using poly-
phosphoric acid. Subsequent acylation using oxalyl
chloride gave the indolylglyoxylyl chlorides 3a–d, which
were aminated using the appropriate amines, for exam-
ple, dihexylamine, di-n-propylamine, diethylamine, or
dimethylamine giving the final N,N-dialkyl-2-phenyl-
indolyl-3-glyoxamides 4a–l in good overall yields.
123I
[
123I]-4c
[
123I]N,N-diethyl-[5-chloro-2-(4-iodophe-
Scheme 2. Preparation of
nyl)indol-3-yl]glyoxylamide. Reagents and conditions: (i) (Sn(CH3)3)2,
Pd(PPh3)4, anhydrous toluene, reflux, 8 h; (ii) Na123I, per-acetic acid,
ethanol, acetic acid.
To prepare the [123I]-radiolabelled analogue of 4c, the
trimethylstannyl precursor 5 was prepared from its cor-
responding bromo derivative 4k, using hexamethylditin
in the presence of a catalytic amount of tetrakis(triphe-
nylphoshine)palladium(0) in anhydrous toluene. The
radioiodination was achieved by an oxidative iodo-
destannylation reaction of 5 and Na123I using per-acetic

3940
T. P. Homes et al. / Bioorg. Med. Chem. 14 (2006) 3938–3946
acid as an oxidising agent (Scheme 2). The radioligand,
123I]N,N-diethyl-[5-chloro-2-(4-iodophenyl)indol-3-yl]-
glyoxylamide was purified and characterised by reverse-
phase HPLC. The radiochemical yield was in the range
of 55–60% (n = 3).
The compounds with a bromo substituent on R3, 4i–l,
had higher PBBS affinities than their respective iodo
analogues 4a–c, and 4e. This suggests that the smaller
the halogen on R3, the better the binding affinity.
Although no direct comparison can be made, com-
pounds reported previously21 with smaller halogen
(fluorine and chlorine) substituents on R3 appeared to
have a higher PBBS binding affinity than the com-
pounds presented here. It was also reported21 that the
optimum alkyl chain lengths (R1 and R2) for com-
pounds with no chloro substituent on R4 were hexyl
groups, whereas with the chloro substituent, the opti-
mum length was propyl. In this work, it was found that
compounds with hexyl groups had the lowest affinities,
with and without a chloro substituent. It was also found
that affinities increased with the addition of a chloro
substituent in compounds containing propyl, ethyl or
methyl groups.21 Compounds 4e and 4l containing hexyl
groups showed higher PBBS affinity than compounds 4a
and 4i with a chloro group in the R4. This suggests that
it is more difficult to accommodate the sterically more
demanding hexyl groups on molecules with R4 = Cl
than in compounds with R4 = H, suggesting different
molecular orientations are possible.
[
3. Results and discussion
The binding affinities (IC50) of the 12 new compounds
for the PBBS were determined by measuring the dis-
placement of [3H]PK11195 bound to rat kidney mito-
chondrial membranes. To determine the selectivity of
the compounds for PBBS versus CBR, CBR binding
affinities were determined using [3H]flumazenil on rat
cortical membranes. The results of the binding affinities
of compounds 4a–l for the PBBS and the CBR are
shown in Table 1. The compounds examined displayed
a medium to high affinity for the PBBS, ranging from
7.86 to 618 nM. All compounds had high selectivity for
PBBS over CBR, with all CBR IC50 > 5000 nM. The
effects of chemical modification to groups R1 to R4
on the binding to PBBS were studied, including the
length of the alkyl chains (R1 and R2), the presence
of a chloro substituent on position 5 of the indole
(R4) and halogen substitution (bromine or iodine) on
the phenyl ring (R3). The most potent ligands for the
PBBS were compounds 4c and 4k, both displaying
IC50 of <10 nM. Decreasing the length of the alkyl
chains from hexyl to propyl to ethyl increased the
binding affinity for the PBBS. However, compounds
with R1 and R2 as methyl groups had a lower
affinity than the compounds with ethyl groups.
To perform radiolabelling, the corresponding trimethyl
stannane 5 was prepared by treatment of the bromo
derivative 4k with hexamethylditin and palladium
tetrakistriphenylphosphine in refluxing toluene. [123I]4c
was prepared by electrophilic iododestannylation in
the presence of per-acetic acid as the oxidant in
55–60% radiochemical yield and above 98% radiochem-
ical purity.
Table 1. Binding affinities for PBBS and CBR, measured and calculated Log P of compounds 4a–l
R1
O
N
O
R2
R4
N
H
R3
b
PBBS IC50 (nM)
c
CBR IC50 (nM)
Compound
R1
R2
R3
R4
Log P
> 6
Calcd log Pa
4a
4b
Hexyl
Propyl
Ethyl
Hexyl
Propyl
Ethyl
I
Cl
Cl
Cl
Cl
H
8.29
5.78
4.81
4.13
7.73
5.22
4.25
3.58
7.76
5.25
4.28
7.20
5.30
169 35
37.4 6.1
8.23 2.2
17.5 4.3
115 22
18786 1634
16156 579
15652 45
10146 1967
13803 137
14662 127
13197 2975
5939 815
13115 3212
10463 2817
11045 1909
16348 716
>1000
I
4.71 0.19
4.00 0.16
3.36 0.13
> 6
4c
4d
I
Methyl
Hexyl
Propyl
Ethyl
Methyl
Hexyl
Propyl
Ethyl
I
4e
I
4f
4g
I
H
4.00 0.16
3.27 0.13
2.69 0.11
> 6
43.6 1.8
19.1 2.5
618 39
I
H
4h
4i
Methyl
Hexyl
Propyl
Ethyl
Methyl
Hexyl
Propyl
Ethyl
I
H
Br
Br
Br
Br
Cl
Cl
Cl
H
138 35
4j
4k
4.56 0.18
3.89 0.15
> 6
16.7 4.7
7.86 1.2
54.7 22.6
3.7 1.2
4l
PK 11195
Hexyl
Hexyl
a Estimated by Chem Draw Ultra.
b The concentration of tested compounds that inhibited [3H]PK11195 binding to rat kidney mitochondrial membranes (IC50) by 50% was determined
with 6 concentrations of the test compounds, each performed in triplicate. IC50 values are means SEM derived from at least 2 independent
experiments.
c The concentration of tested compounds that inhibited [3H]flumazenil binding to rat cortex membranes (IC50) by 50% was determined with 6
concentrations of the test compounds, each performed in triplicate. IC50 values are means SEM derived from at least 2 independent experiments.

T. P. Homes et al. / Bioorg. Med. Chem. 14 (2006) 3938–3946
3941
Lipophilicity measurements of all compounds were as-
sessed in order to explain their in vivo properties. As
expected the presence of bulky alkyl groups (R1 and
R2) greatly influenced the overall lipophilicity of the
molecules resulting in compounds 4a, 4e, 4i and 4l hav-
ing log P values greater than 6 and molecules with pro-
pyl groups 4b, 4f and 4j greater than 4. In addition,
incorporating a chloro substituent on R4 increased the
log P values from 3.27 in 4g and 2.69 in 4h to 4.0 in
4c and 3.36 in 4d, respectively. However, the presence
of small alkyl groups such as ethyl and methyl in the
R1 and R2 positions and hydrogen in the R4 positions
can yield compounds with log P values between 2 and 4.
[3H]PK11195 and [3H]flumazenil were purchased from
Perkin-Elmer Life Sciences (Boston, MA, USA).
PK11195 and flumazenil were purchased from Sigma-
RBI. For binding studies, male Sprague–Dawley rats
were purchased from Animal Resources Centre (Perth,
WA, Australia). All procedures were carried out in com-
pliance with Australian laws governing animal
experimentation.
5.1. 4-Iodoacetophenone 4-chlorophenylhydrazone (1a)
A mixture of 4-iodoacetophenone (4.15 g, 16.9 mmol),
4-chlorophenylhydrazine
hydrochloride
(3.00 g,
16.8 mmol) and a few drops of glacial acetic acid in
ethanol (40 mL) was stirred and heated at reflux for
45 min. A precipitate that formed was filtered and
washed with dilute HCl followed by cold 95% ethanol
(10 mL). The product was recrystallised in ethanol to
yield 1a (3.34 g, 54%) as yellow crystals: mp 130–
4. Conclusion
A series of N,N-dialkyl-[2-(40-iodo- and 40-bromo-phenyl-
indol-3-yl]glyoxylamides) 4a–l were synthesised and
their binding affinity for the PBBS and CBR was deter-
mined using [3H]PK11195 and [3H]flumazenil on rat
kidney mitochondrial and cortical membranes, respec-
tively. The brominated and iodinated compounds 4c
and 4k bearing a chloro substituent on R4 and ethyl
groups at R1 and R2 displayed the highest affinity for
the PBBS. All compounds displayed low affinity for
the CBR (IC50 > 5000 nM). Compound 4c was radiola-
belled with iodine-123 by electrophilic iododestannyla-
tion using per-acetic acid as the oxidant in 55–60%
radiochemical yield and >98% radiochemical purity.
1
132 ꢁC. H NMR (CDCl3) d 2.21 (s, 3H, CH3), 7.11
(d, 2H, J = 8.9, 2ArH), 7.24 (d, 2H, J = 8.9, 2ArH),
7.34 (bs, 1H, NH), 7.51 (d, 2H, J = 8.6, 2ArH), 7.71
(d, 2H, J = 8.6, 2ArH). 13C NMR (CDCl3) d 12.0,
94.2, 114.8, 125.4, 127.7, 129.6, 137.8, 138.8, 141.0,
144.0. MS (EI) m/z 370 (M+, 33%). Anal. Calcd for
C14H12ClIN2: C, 45.37; H, 3.26; N, 7.56. Found: C,
45.67; H, 3.37; N, 7.47.
5.2. 4-Iodoacetophenone phenylhydrazone (1b)
In similar manner, 4-iodoacetophenone (5.00 g,
20.3 mmol) and phenylhydrazine (2.0 mL, 20.3 mmol)
in ethanol (30 mL) gave 1b (4.38 g, 64%) as orange crys-
tals.1H NMR (CDCl3) d 2.19 (s, 3H, CH3), 6.89 (t, 1H,
J = 7.2, ArH4), 7.16 (d, 2H, J = 7.8, 2ArH), 7.28 (m,
2H, 2ArH), 7.52 (d, 2H, J = 8.6, 2ArH), 7.68 (d, 2H,
J = 8.6, 2ArH). MS (EI) m/z 336 (M+, 100%).
5. Experimental
Melting points were determined on a Gallenkamp melt-
ing point apparatus and are uncorrected. Elemental anal-
yses were performed on a Carlo Erba 1106 Elemental
analyser at the Campbell Microanalytical Laboratory,
University of Otago (Dunedin, New Zealand), and their
5.3. 4-Bromoacetophenone 4-chlorophenylhydrazone (1c)
1
results are within 0.4% of theoretical values. H and
13C nuclear magnetic resonance (NMR) spectra were
recorded using a Bruker 400 MHz spectrometer. Chemi-
cal shifts (d) are expressed in ppm, and the coupling
constants (J) in Hz. Signals are recorded as singlet (s),
doublet (d), triplet (t), quartet (q) and multiplet (m). Mass
spectra (MS) were obtained using either a VG Quattro
triple quadrupole system or an Autospec high-resolution
mass spectrometer. Analytical thin layer chromatogra-
phy (TLC) was performed on Merck Kieselgel 60 F254
precoated polyester plates with a thickness of 250 lm.
Column chromatography was performed on Merck
Keiselgel 60 (220–440 mesh). Reagents and solvents were
purchased from commercial sources and were used
without further purification. Petroleum ether of boiling
point range 30–60 ꢁC was used. Yields refer to purified
products and are not optimised. Chromatographic
separation of radiolabelled product was carried out on
a Phenomenex bondclone semi-preparative RP C-18
column (10 l, 7.8 · 300 mm) using a Waters 510 pump,
an Activon Linear UV detector set at 254 nm, and an
in-line NaI-Berthold radioactivity detector. Carrier free
Na123I in 0.1 M NaOH was obtained from Australian
Radioisotopes and Industrials (ARI), Australia.
In a similar manner, 4-bromoacetophenone (6.89 g,
34.6 mmol) and 4-chlorophenylhydrazine hydrochloride
(6.20 g, 34.6 mmol) in ethanol (60 mL) gave 1c (8.38 g,
75%) as pale yellow crystals: mp 132–134 ꢁC. 1H
NMR (CDCl3) d 2.20 (s, 3H, CH3), 7.09 (d, 2H,
J = 8.9, 2ArH), 7.23 (d, 2H, J = 8.9, 2ArH), 7.33 (bs,
1H, NH), 7.49 (d, 2H, J = 8.7, 2ArH), 7.64 (d, 2H,
J = 8.7, 2ArH). 13C NMR (CDCl3) d 12.1, 114.8,
122.6, 125.4, 127.5, 129.6, 130.2, 131.8, 138.1, 144.0.
MS (EI) m/z 324 (M+, 74%), 126 (M+ꢀ198, 100%).
Anal. Calcd for C14H12N2ClBr: C, 51.96; H, 3.74; N,
8.66. Found: C, 52.07; H, 3.59; N, 8.88.
5.4. 5-Chloro-2-(4-iodophenyl)indole (2a)
A mixture of 1a (3.10 g, 8.4 mmol) and polyphosphoric
acid (20 g) was stirred for 30 min at 110 ꢁC. Ice water
(250 mL) was added and the mixture was stirred. The
precipitate was filtered, washed with water and recrys-
tallised from 95% ethanol, to yield 2a (1.66 g, 56%) as
1
a white solid: mp 210–211 ꢁC. H NMR (DMSO-d6) d
6.92 (s, 1H, H3), 7.10 (dd, 1H, J6,7 = 8.6, J6,4 = 2.0,
H6), 7.39 (d, 1H, J7,6 = 8.6, H7), 7.57, (d, 1H,

3942
T. P. Homes et al. / Bioorg. Med. Chem. 14 (2006) 3938–3946
J4,6 = 1.9, H4), 7.66 (d, 2H, J = 8.4, 2ArH), 7.83 (d, 2H,
J = 8.4, 2ArH), 11.77 (s, 1H, NH). 13C NMR (DMSO-
d6) d 95.4, 100.6, 114.5, 120.9, 123.5, 125.7, 128.8,
131.3, 132.9, 137.3, 139.4, 139.9. MS (CI) m/z 354
(M+, 34%), 228 (M+ꢀ126, 100%). Anal. Calcd for
C14H9ClIN: C, 47.56; H, 2.57; N, 3.96. Found: C,
47.56; H, 2.36; N, 4.18.
H6), 7.38 (d, 2H, J = 8.1, 2· ArH), 7.54 (d, 1H,
J7,6 = 8.6, H7), 7.89 (d, 2H, J = 8.1, 2· ArH), 8.08 (s,
1H, H4).
5.9. [2-(4-Iodophenyl)indol-3-yl]glyoxylyl chloride (3b)
Treatment of 2b (0.39 g, 1.22 mmol) and oxalyl chloride
(0.15 mL, 1.70 mmol) in diethyl ether (6.5 mL) for 1 h as
1
5.5. 2-(4-Iodophenyl)indole (2b)23
above gave 3b (0.34 g, 68%). H NMR (DMSO-d6) d
7.25–7.35 (m, 2H, H5 and H6), 7.39 (d, 2H, J = 8.2,
2· ArH), 7.52 (d, 1H, J7,6 = 7.6, H7), 7.89 (d, 2H,
J = 8.2, 2· ArH), 8.09 (d, 1H, J4,5 = 7.4, H4), 12.83 (s,
1H, NH).
Treatment of a mixture of 1b (3.09 g, 9.19 mmol) and
polyphosphoric acid (15 g) at 60–70 ꢁC as above gave
1
2b (1.70 g, 58%) as a white solid. H NMR (CDCl3) d
6.82 (s, 1H, H3), 7.12 (t, 1H, H5 or H6), 7.21 (t, 1H,
H6 or H5), 7.38 (d, 3H, 2ArH and H7), 7.62 (d, 2H,
J = 7.8, H4), 7.76 (d, 2H, J = 10.8, 2ArH), 8.27 (bs,
1H, NH). 13C NMR (DMSO-d6) d 94.1, 100.5, 112.5,
120.7, 121.4, 123.1, 128.1, 129.7, 132.9, 137.7, 138.4,
138.8. MS (EI) m/z 319 (M+, 100%).
5.10. [2-(4-Bromophenyl)-5-chloroindol-3-yl]glyoxylyl
chloride (3c)
Reaction of 2c (1.55 g, 5.06 mmol) and oxalyl chloride
(0.62 mL, 7.08 mmol) in diethyl ether (14 mL) for 4 h
gave 3c (1.96 g, 98%). 1H NMR (DMSO-d6) d 7.34
(dd, 1H, J6,7 = 8.6, J6,4 = 2.1, H6), 7.38 (d, 2H, J = 8.3,
2· ArH), 7.54 (d, 1H, J7,6 = 8.6, H7), 7.90 (d, 2H,
J = 8.3, 2· ArH), 8.08 (d, 1H, J4,6 = 2.0, H4), 12.84 (s,
1H, NH).
5.6. 2-(4-Bromophenyl)-5-chloroindole (2c)
Treatment of a mixture of 1c (5.12 g, 15.8 mmol) and
polyphosphoric acid (30 g) as above gave 2c (3.35 g,
1
69%) as a pale yellow solid: mp. 206–207 ꢁC. H NMR
(DMSO-d6)
d
6.91 (s, 1H, H3), 7.11 (dd, 1H,
5.11. [2-(4-Bromophenyl)indol-3-yl]glyoxylyl chloride (3d)
J6,7 = 8.6, J6,4 = 2.0, H6), 7.40 (d, 1H, J7,6 = 8.6, H7),
7.57 (d, 1H, J4,6 = 1.9, H4), 7.66 (d, 2H, J = 8.5,
2ArH), 7.80 (d, 2H, J = 8.5, 2ArH), 11.79 (s, 1H,
NH). 13C NMR (DMSO-d6) d 99.0, 112.8, 119.2,
120.8, 121.8, 124.1, 127.1, 129.7, 130.9, 131.9, 135.7,
138.1. MS (CI) m/z 308 (M+,79%), 228 (M+ꢀ79,
100%). Anal. Calcd for C14H9BrClN: C, 54.85; H,
2.96; N, 4.57. Found: C, 54.93; H, 3.11; N, 4.50.
Reaction of 2d (2.00 g, 7.35 mmol) and oxalyl chloride
(0.90 mL, 10.3 mmol) in diethyl ether (20 mL) for 1 h
gave 3d (1.75 g, 66%). H NMR (CDCl3) d 7.37–7.41
(m, 2H, H5 and H6), 7.42 (d, 2H, J = 8.5, 2· ArH),
7.44–7.49 (m, 1H, H7), 7.66 (d, 2H, J = 8.5, 2· ArH),
8.24–8.29 (m, 1H, H4), 8.92 (bs, 1H, NH).
1
5.12. N,N-Dihexyl-[5-chloro-2-(4-iodophenyl)indol-3-
yl]glyoxylamide (4a)
5.7. 2-(4-Bromophenyl)indole (2d)22
To
a
mixture of 4-bromoacetophenone (6.89 g,
To a solution of the chloride 3a (0.30 g, 0.67 mmol) and
triethylamine (0.11 mL, 0.81 mmol) in anhydrous tolu-
ene (30 mL) at 0 ꢁC was added dropwise a solution of
dihexylamine (0.17 mL, 0.74 mmol) in anhydrous tolu-
ene (20 mL). The solution was stirred at room tempera-
ture for 24 h. The solution was filtered and the filtrate
was washed sequentially with 0.5 M HCl, saturated
NaHCO3 and water. The combined organic extracts
were dried with MgSO4, filtered, and the solvent was
evaporated. The crude product was purified by column
chromatography (methanol/chloroform, 1:19 v/v) and
then recrystallised (ethyl acetate/petroleum ether, 1:9 v/
v) to give 4a (0.12 g, 30%) as a white solid: mp 108–
110 ꢁC. 1H NMR (DMSO-d6) d 0.75 (t, 3H, CH3),
0.88 (t, 3H, CH3), 1.00–1.20 (m, 10H, 5CH2), 1.21–
1.35 (m, 4H, 2CH2), 1.36–1.46 (m, 2H, CH2), 2.94–
3.08 (m, 4H, 2NCH2), 7.32 (dd, 1H, J6,7 = 8.7,
J6,4 = 1.8, H6), 7.37 (d, 2H, J = 8.1, 2· ArH), 7.50 (d,
1H, J7,6 = 8.7, H7), 7.88 (d, 2H, J = 8.1, 2· ArH), 8.04
(s, 1H, H4), 12.68 (bs, 1H, NH). 13C NMR (DMSO-
d6) d 15.4, 15.6, 23.5, 23.7, 27.2, 27.9, 28.1, 29.3, 32.3,
32.8, 45.4, 48.9, 98.7, 111.0, 115.5, 121.7, 125.3, 128.9,
129.5, 131.4, 133.5, 136.0, 138.6, 148.9, 168.6, 188.6.
MS (EI) m/z 592 (M+, 3%), 380 (M+ꢀ212, 100%).
HRMS-ES calculated for C28H35ClIN2O2: 593.1432,
found 593.1470. Anal. Calcd for C28H34ClIN2O2: C,
34.6 mmol) and phenylhydrazine (3.4 mL, 34.6 mmol)
was added polyphosphoric acid (40 g). The reaction
mixture was heated at 110 ꢁC, stirring occasionally until
the mixture turned a deep reddish brown colour. The
reaction mixture was worked up the same as 2a to yield
1
2d (5.41 g, 58%) as a white solid. H NMR (CDCl3) d
6.82 (s, 1H, H3), 7.13 (t, 1H, J = 7.9, H5 or H6), 7.21
(t, 1H, J = 8.2, H6 or H5), 7.39 (d, 1H, J = 8.0, H7),
7.51 (d, 2H, J = 8.7, 2ArH), 7.56 (d, 2H, J = 8.7,
2ArH), 7.62 (d, 1H, J = 8.0, H4), 8.27 (bs, 1H, NH).
MS (EI) m/z 271 (79BrM+, 100%), 273 (81BrM+, 98%).
HRMS-EI+ calculated for C14H10NBr: 270.9997, found
270.9995.
5.8. [5-Chloro-2-(4-iodophenyl)indol-3-yl]glyoxylyl
chloride (3a)
To a partially dissolved solution of indole 2a (0.65 g,
1.84 mmol) in anhydrous diethyl ether at 0 ꢁC was
added dropwise oxalyl chloride (0.22 mL, 2.57 mmol)
and the mixture was stirred for 6 h at room temperature.
The precipitate was collected by filtration and washed
with a small portion of diethyl ether to yield 3a
(0.72 g, 88%), which was directly used to synthesise
1
4a–d. H NMR (DMSO-d6) d 7.33 (d, 1H, J6,7 = 8.6,

T. P. Homes et al. / Bioorg. Med. Chem. 14 (2006) 3938–3946
3943
56.72; H, 5.78; N, 4.72. Found: C, 56.49; H, 5.75; N,
4.72.
lated for C18H15ClIN2O2: 452.9867, found 452.9872.
Anal. Calcd for C18H14ClIN2O2: C, 47.76; H, 3.12; N,
6.19. Found: C, 47.54; H, 3.39; N, 5.89.
5.13. N,N-Di-n-propyl-[5-chloro-2-(4-iodophenyl)indol-3-
yl]glyoxylamide (4b)
5.16. N,N-Dihexyl-[2-(4-iodophenyl)indol-3-yl]glyoxyl-
amide (4e)
Reaction of 3a (0.72 g, 1.62 mmol) and di-n-propyl-
amine (0.25 mL, 1.78 mmol) for 7 h as above gave after
trituration with petroleum ether, 4b (0.47 g, 57%) as an
off-white solid: mp 148–150 ꢁC. H NMR (DMSO-d6) d
Reaction of 3b (0.23 g, 0.56 mmol) and dihexylamine
(0.15 mL, 0.62 mmol) for 18 h followed by column chro-
matography (ethyl acetate/petroleum ether, 2:3 v/v) and
recrystallisation (ethanol/water) gave 4e (0.10 g, 32%) as
1
0.69 (t, 3H, J = 7.4, CH3), 0.78 (t, 3H, J = 7.4, CH3),
1.12–1.28 (m, 2H, CH2), 1.40–1.50 (m, 2H, CH2), 2.95
(t, 2H, J = 7.8, NCH2), 3.01 (t, 2H, J = 7.7, NCH2),
7.31 (dd, 1H, J6,7 = 8.6, J6,4 = 2.1, H6), 7.35 (d, 2H,
J = 8.3, 2· ArH), 7.51 (d, 1H, J7,6 = 8.6, H7), 7.87 (d,
2H, J = 8.3, 2· ArH), 8.02 (d, 1H, J4,6 = 1.9, H4). 13C
NMR (DMSO-d6) d 12.5, 13.1, 21.6, 22.8, 47.2, 50.8,
98.6, 111.1, 115.5, 121.6, 125.3, 128.9, 129.5, 131.4,
133.4, 136.0, 138.6, 149.0, 168.8, 188.5. MS (ES) m/z
509 (M+1, 88%), 106 (100%). HRMS-ES calculated
for C22H23ClIN2O2: 509.0493, found 509.0524. Anal.
Calcd for C22H22ClIN2O2: C, 51.94; H, 4.36; N, 5.51.
Found: C, 52.14; H, 4.58; N, 5.53.
1
a white solid: mp 120–122 ꢁC. H NMR (DMSO-d6) d
0.74 (t, 3H, J = 7.0, CH3), 0.84 (t, 3H, J = 6.9, CH3),
1.00–1.45 (m, 16H, 8CH2), 2.95–3.07 (m, 4H, 2NCH2),
7.21–7.31 (m, 2H, H5 and H6), 7.37 (d, 2H, J = 8.3,
2· ArH), 7.48 (d, 1H, J = 7.7, H7), 7.87 (d, 2H,
J = 8.3, 2· ArH), 8.04 (d, 1H, J = 7.7, H4). 13C NMR
(DMSO-d6) d 13.8, 14.0, 21.8, 22.1, 25.6, 26.2, 26.4,
27.5, 30.6, 31.1, 43.7, 47.3, 96.7, 109.8, 112.1, 120.9,
122.5, 123.6, 126.7, 130.2, 131.9, 135.8, 136.8, 146.0,
167.3, 187.1. MS (EI) m/z 558 (M+, 4%), 346
(M+ꢀ212,
100%).
HRMS-EI
C28H35N2O2I: 558.1743, found 558.1747.
calculated
for
5.14. N,N-Diethyl-[5-chloro-2-(4-iodophenyl)indol-3-yl-
glyoxylamide (4c)
5.17. N,N-Di-n-propyl-[2-(4-iodophenyl)indol-3-yl]gly-
oxylamide (4f)
Reaction of 3a (0.36 g, 0.80 mmol) and diethylamine
(0.09 mL, 0.88 mmol) for 19 h yielded after column
chromatography (ethyl acetate/petroleum ether, 1:1
v/v) 4c (0.14 g, 36%) as colourless crystals: mp 221–
222 ꢁC. 1H NMR (DMSO-d6) d 0.83 (t, 3H, J = 7.1,
CH3), 1.02 (t, 3H, J = 7.0, CH3), 3.07 (q, 2H, J = 7.1,
CH2), 3.15 (q, 2H, J = 7.0, CH2), 7.32 (dd, 1H,
J6,7 = 8.6, J6,4 = 2.1, H6), 7.37 (d, 2H, J = 8.3, 2·
ArH), 7.51 (d, 1H, J7,6 = 8.6, H7), 7.88 (d, 2H, J = 8.3,
2· ArH), 8.05 (d, 1H, J4,6 = 2.0, H4). 13C NMR
(DMSO-d6) d 12.5, 14.1, 38.4, 42.2, 97.4, 109.8, 114.3,
120.5, 124.1, 127.7, 128.4, 130.3, 132.3, 134.8, 137.3,
147.9, 167.1, 187.4. MS (ES) m/z 478 (Mꢀ1, 100%).
HRMS-ES calculated for C20H19ClIN2O2: 481.0180,
found 481.0180. Anal. Calcd for C20H18ClIN2O2: C,
49.97; H, 3.77; N, 5.83. Found: C, 50.27; H, 4.05; N,
5.85.
Reaction of 3b (0.23 g, 0.56 mmol) and di-n-propyl-
amine (0.09 mL, 0.62 mmol) for 20 h, followed by col-
umn chromatography (ethyl acetate/petroleum ether,
1:1 v/v) and trituration with ethyl acetate/petroleum
ether, 3:7, v/v, gave 4f (58.2 mg, 22%) as an off-white
1
solid: mp 159–161 ꢁC. H NMR (DMSO-d6) d 0.69 (t,
3H, J = 7.4, CH3), 0.79 (t, 3H, J = 7.4, CH3), 1.13–
1.25 (m, 2H, CH2), 1.41–1.52 (m, 2H, CH2), 2.96 (t,
2H, J = 7.9, NCH2), 3.03 (t, 2H, J = 7.7, NCH2),
7.21–7.33 (m, 2H, H5 and H6), 7.37 (d, 2H,
J = 8.4, 2· ArH), 7.48 (d, 1H, J = 7.3, H7), 7.88 (d,
2H, J = 8.4, 2· ArH), 8.03 (d, 1H, J = 7.3, H4). 13C
NMR (DMSO-d6) d 11.9, 12.4, 20.9, 22.2, 46.5,
50.2, 97.6, 110.8, 113.1, 121.8, 123.5, 124.5, 127.6,
131.3, 132.9, 136.8, 137.8, 147.0, 168.4, 188.0. MS
(EI) m/z 474 (M+, 6%), 346 (M+ꢀ128, 100%).
HRMS-EI calculated for C22H23N2 O2I: 474.0804,
found 474.0795.
5.15. N,N-Dimethyl-[5-chloro-2-(4-iodophenyl)indol-3-
yl]glyoxylamide (4d)
5.18. N,N-Diethyl-[2-(4-iodophenyl)indol-3-yl]glyoxyl-
amide (4g)
Dimethylamine (30 mL, 40% wt/vol in water) was gently
heated and under constant N2 pressure bubbled through
NaOH pellets into a solution of 3a (0.65 g, 1.46 mmol)
in toluene. After purification by column chromatogra-
phy (ethyl acetate/petroleum ether, 1:1 v/v) and tritur-
ation with ethyl acetate/petroleum ether, 3:7 v/v, 4d
(65 mg, 10%) was obtained as a solid: mp 238 ꢁC
(decomposed). 1H NMR (DMSO-d6) d 2.45 (s, 3H,
NCH3), 2.79 (s, 3H, NCH3), 7.32 (d, 2H, J = 8.3, 2·
ArH), 7.33 (dd, 1H, J6,7 = 8.6, H6), 7.52 (d, 1H,
J7,6 = 8.6, H7), 7.91 (d, 2H, J = 8.3, 2· ArH), 8.11 (d,
1H, J = 2.0, H4). 13C NMR (DMSO-d6) d 33.7, 37.4,
97.9, 110.6, 114.8, 121.1, 124.7, 128.3, 128.8, 130.6,
132.5, 135.3, 137.7, 148.8, 167.5, 188.1. MS (ES) m/z
453 (M+1, 21%), 338 (Mꢀ114, 100%). HRMS-ES calcu-
Reaction of 3b (0.14 g, 0.34 mmol) and diethylamine
(0.05 mL, 0.47 mmol) for 3 h gave after recrystallisation
(ethyl acetate/petroleum ether, 1:1 v/v) 4g (60 mg, 40%)
as off-white crystals: mp 169–171 ꢁC. 1H NMR (DMSO-
d6) d 0.85 (t, 3H, J = 7.1, CH3), 1.02 (t, 3H, J = 7.0,
CH3), 3.07 (q, 2H, J = 7.1, NCH2), 3.15 (q, 2H,
J = 7.0, NCH2), 7.22–7.32 (m, 2H, H5 and H6), 7.37
(d, 2H, J = 8.3, 2· ArH), 7.49 (d, 1H, J = 7.3, H7),
7.87 (d, 2H, J = 8.3, 2· ArH), 8.06 (d, 1H, J = 7.2,
H4). 13C NMR (DMSO-d6) d 13.8, 15.3, 39.5, 43.5,
98.3, 111.5, 113.8, 122.5, 124.2, 125.3, 128.4, 131.9,
133.6, 137.3, 138.4, 147.7, 168.7, 188.8. MS (ES) m/z
445 (Mꢀ1, 100%). HRMS-EI calculated for
C20H19IN2O2: 446.0491, found 446.0489. Anal. Calcd

3944
T. P. Homes et al. / Bioorg. Med. Chem. 14 (2006) 3938–3946
for C20H19IN2O2: C, 53.83; H, 4.29; N, 6.28. Found: C,
53.50; H, 4.41; N, 6.11.
127.9, 128.4, 130.2, 131.7, 132.5, 135.0, 147.8, 167.8,
187.4. MS (ES) m/z 463 (81Br M+1, 61%), 106
(Mꢀ357, 100%). HRMS-ES calculated for C22H23N2
O2ClBr 461.0631, found 461.0649. Anal. Calcd for
C22H22BrClN2O2: C, 57.22; H, 4.80; N, 6.07. Found:
C, 57.33; H, 4.89; N, 6.01.
5.19. N,N-Dimethyl-[2-(4-iodophenyl)indol-3-yl]glyoxyl-
amide (4h)
Dimethylamine (30 mL, 40% wt/vol in water) was gently
heated and under constant N2 pressure bubbled through
NaOH pellets into a solution of 3b (0.30 g, 0.73 mmol)
in toluene. After column chromatography (ethyl ace-
tate/petroleum ether, 1:1 v/v), 4h was obtained (14 mg,
5%) as an off-white solid: mp 231–232 ꢁC. 1H NMR
(DMSO-d6) d 2.49 (s, 3H, NCH3), 2.79 (s, 3H, NCH3),
7.20–7.30 (m, 2H, H5 and H6), 7.34 (d, 2H, J = 8.1,
2· ArH), 7.48 (d, 1H, J = 7.0, H7), 7.89 (d, 2H,
J = 8.3, 2· ArH), 8.07 (d, 1H, H4). 13C NMR
(DMSO-d6) d 34.4, 38.1, 98.0, 111.3, 114.0, 122.5,
124.3, 125.2, 128.2, 130.5, 133.3, 138.2, 168.8, 187.2.
MS (EI) m/z 418 (M+, 6%), 346 (M+ꢀ72, 100%).
HRMS-EI calculated for C18H15N2O2I: 418.0178, found
418.0161. Anal. Calcd for C18H15IN2O. H2O: C, 49.56;
H, 3.93; N, 6.42. Found: C, 49.70; H, 3.46; N, 6.29
5.22. N,N-Diethyl-[2-(4-bromophenyl)-5-chloroindol-3-
ylglyoxylamide (4k)
To a solution 3c (0.72 g, 1.81 mmol) in anhydrous tolu-
ene (40 mL) at 0 ꢁC was added dropwise a solution of
triethylamine (0.30 mL, 2.18 mmol) and diethylamine
(0.21 mL, 1.99 mmol) in anhydrous toluene (10 mL).
The reaction mixture was stirred at room temperature
for 18 h. A white precipitate that formed was filtered,
washed with water (25 mL) and extracted into dichloro-
methane (2· 40 mL). The combined organic extracts
were dried with MgSO4, filtered, and the solvent was
evaporated in vacuo. After recrystallisation (ethyl ace-
tate/petroleum ether, 1:1 v/v), 4k (0.462 g, 59%) was ob-
tained as a white solid: mp 216–217 ꢁC. 1H NMR
(DMSO-d6) d 0.84 (t, 3H, J = 7.1, CH3), 1.02 (t, 3H,
J = 7.0, CH3), 3.07 (q, 2H, J = 6.9, NCH2), 3.15 (q,
2H, J = 6.8, NCH2), 7.32 (d, 1H, J = 8.6, H6), 7.47–
7.56 (m, 3H, 2· ArH and H7), 7.72 (d, 2H, 2· ArH),
8.05 (s, 1H, H4). 13C NMR (DMSO-d6) d 13.0, 14.6,
38.9, 42.7, 110.4, 114.9, 121.0, 124.5, 124.6, 128.1,
128.9, 130.1, 132.0, 132.9, 135.3, 148.5, 167.7, 188.0.
MS (EI) m/z 434 (M+, 4%), 334 (M+ꢀ100, 100%). Anal.
Calcd for C20H18BrClN2O2: C, 55.38; H, 4.18; N, 6.46.
Found: C, 55.37; H, 4.36; N, 6.40.
5.20. N,N-Dihexyl-[2-(4-bromophenyl)-5-chloroindol-3-
yl]glyoxylamide (4i)
Reaction of 3c (0.46 g, 1.16 mmol) and dihexylamine
(0.30 mL, 1.27 mmol) for 7 h, followed by purification
by column chromatography (ethyl acetate/petroleum
ether, 2:3 v/v) and recrystallisation (ethyl acetate/petro-
leum ether, 1:9 v/v), gave 4i (0.20 g, 32%) as a white
solid: mp 103–105 ꢁC. 1H NMR (DMSO-d6) d 0.77
(t, 3H, J = 7.0, CH3), 0.87 (t, 3H, J = 6.8, CH3),
1.00–1.46 (m, 16H, 8CH2), 2.95–3.09 (m, 4H,
2NCH2), 7.32 (dd, 1H, J6,7 = 8.6, J6,4 = 2.0, H6), 7.51
(d, 1H, J7,6 = 8.7, H7), 7.54 (d, 2H, J = 8.4, 2· ArH)
7.72 (d, 2H, J = 8.4, 2· ArH), 8.03 (s, 1H, H4). 13C
NMR (DMSO-d6) d 14.7, 14.9, 22.8, 23.0, 26.5, 27.1,
27.4, 28.6, 31.6, 32.0, 44.7, 48.2, 110.4, 114.8, 121.0,
124.5, 124.6, 128.1, 128.7, 130.4, 132.0, 132.9, 135.3,
148.0, 167.9, 187.8. MS (EI) m/z 546 (M+, 1%), 334
5.23. N,N-Dihexyl-[2-(4-bromophenyl)indol-3-yl]gly-
oxylamide (4l)
Reaction of 3d (0.96 g, 2.65 mmol) and dihexylamine
(0.68 mL, 2.91 mmol) for 24 h gave after purification
by column chromatography (methanol/dichloro-
methane, 1:19 v/v) and recrystallisation (ethanol/water),
1
4l (0.62 g, 46%) as a white solid: mp 106–110 ꢁC. H
(M+ꢀ212,
100%).
HRMS-EI
calculated
for
NMR (DMSO-d6) d 0.74 (t, 3 H, J = 7.0, CH3), 0.88
(t, 3H, J = 6.9, CH3), 1.00–1.20 (m, 10H, 5CH2), 1.20–
1.30 (m, 4H, 2CH2), 1.35–1.47 (m, 2H, CH2), 2.95–
3.08 (m, 4H, 2NCH2), 7.21–7.32 (m, 2H, H5 and H6),
7.48 (d, 1H, J = 7.6, H7), 7.53 (d, 2H, J = 8.4, 2·
ArH), 7.70 (d, 2H, J = 8.4, 2· ArH), 8.05 (d, 1H,
J = 7.8, H4), 12.49 (s, 1H, NH). 13C NMR (DMSO-
d6) d 15.4, 15.6, 23.5, 23.7, 27.2, 27.8, 28.1, 29.4, 32.3,
32.7, 45.4, 49.0, 111.5, 113.8, 122.6, 124.2, 124.9,
125.3, 128.3, 131.6, 132.6, 133.7, 137.4, 147.4, 168.9,
188.8. MS (CI) m/z 511 (79Br M+, 4%), 513 (81Br M+,
3%), 298 (79Br M+ꢀ213, 100%), 300 (81Br M+ꢀ213,
80%). HRMS-EI calculated for C28H35BrN2O2:
510.1882, found 510.1874. Anal. Calcd for
C28H35BrN2O2: C, 65.75; H, 6.90; N, 5.48. Found: C,
65.64; H, 6.78; N, 5.49.
C28H3481Br37ClN2O2: 548.1442, found 548.1442. Anal.
Calcd for C28H34BrClN2O2: C, 61.60; H, 6.28; N,
5.13. Found: C, 61.35; H, 6.02; N, 5.06.
5.21. N,N-Di-n-propyl-[2-(4-bromophenyl)-5-chloroindol-
3-yl]glyoxylamide (4j)
Reaction of 3c (0.41 g, 1.03 mmol) and di-n-propyl-
amine (0.16 mL, 1.13 mmol) for 24 h, followed by puri-
fication by column chromatography (ethyl acetate/
petroleum ether, 1:1 v/v) and recrystallisation (diethyl
ether/petroleum ether), gave 4j (0.25 g, 53%) as a white
1
solid: mp 128–129 ꢁC. H NMR (DMSO-d6) d 0.69 (t,
3H, J = 7.4, CH3), 0.77 (t, 3H, J = 7.4, CH3), 1.15–
1.25 (q, 2H, J = 7.8, CH2), 1.40–1.50 (q, 2H, J = 7.6,
CH2), 2.96 (t, 2H, J = 7.8, NCH2), 3.02 (t, 2H, J = 7.7,
NCH2), 7.31 (dd, 1H, J6,7 = 8.6, J6,4 = 2.1, H6), 7.51
(d, 1H, J7,6 = 8.7, H7), 7.52 (d, 2H, J = 8.5, 2· ArH),
7.70 (d, 2H, J = 8.5, 2· ArH), 8.00 (dd, 1H,
J4,6 = 1.74, H4). 13C NMR (DMSO-d6) d 11.5, 12.0,
20.6, 21.8, 46.2, 49.9, 110.1, 114.6, 120.6, 124.2, 124.3,
5.24. N,N-Diethyl-5-chloro-[2-(4-trimethylstannyl-
phenyl)indol-3-yl]glyoxylamide (5)
To a solution of 4k (112 mg, 0.259 mmol) in anhydrous
toluene (8 mL) were added hexamethylditin (230 lg,

T. P. Homes et al. / Bioorg. Med. Chem. 14 (2006) 3938–3946
3945
0.702 mmol) and a catalytic amount of Pd(0)(PPh3)4.
The reaction mixture was heated at reflux under a nitro-
gen atmosphere for 8 h. The reaction mixture was
passed through Celite and the solvent evaporated. The
product was purified by column chromatography (ethyl
acetate/petroleum ether, 1:1 v/v) to yield [123I]4c as a
50 mM Tris–HCl at pH 7.4 in the presence of
[3H]PK11195 (2 nM) at a final protein concentration
of 250 lg/mL in a final volume of 0.5 mL. Non-specific
binding was determined using PK11195 (10 lM), a
selective PBBS ligand. Samples were incubated for
1 h at 4 ꢁC and then filtered under reduced pressure
on glass fibre filters (Whatman GF/B) pre-soaked in
50 mM Tris–HCl. After incubation, filters were imme-
diately washed 4 times with 4 mL ice-cold Tris–HCl
buffer and the radioactivity remaining on the filters
was measured in a b-scintillation counter (Packard).
IC50 values were calculated using an iterative non-
linear least-squared curve fitting program (Kell
Radioligand).
1
white solid (42.7 mg, 32%). H NMR (CD3OD) d 0.36
(s, 9H, Sn(CH3)3), 0.79 (t, 3H, J = 7.2, CH3), 1.15 (t,
3H, J = 7.2, CH3), 3.07 (q, 2H, J = 7.2, NCH2), 3.24
(q, 2H, J = 7.2, NCH2), 7.32 (dd, 1H, J6,7 = 8.4,
J6,4 = 2.0, H6), 7.47 (d, 1H, J6,7 = 8.4, H7), 7.54 (d,
2H, J = 8.0, 2ArH), 7.65 (d, 2H, J = 8.0, 2ArH), 8.27
(d, 1H, J4,6 = 2.0, H4). MS-ES m/z 517 (Mꢀ1, 70%).
HRMS-ES calculated for C23H28N2 O2Cl116Sn:
515.0857, found 515.0903.
5.27.2. Central benzodiazepine receptor. Cortical mem-
branes were extracted from male Sprague–Dawley rats.
The cortex was removed, rinsed with ice-cold 50 mM
Tris–HCl buffer (pH 7.4), weighed and finely cut. After
the addition of 20 volumes of ice-cold buffer, the prepa-
ration was homogenised and the suspension was centri-
fuged at 20,000g for 20 min at 4 ꢁC. The pellets were
resuspended in buffer and centrifuged as above. The pel-
let was resuspended in 50 mM Tris–HCl, 0.32 M sucrose
buffer (pH 7.4) to achieve a protein concentration of
4 mg/mL, and the membranes were stored at ꢀ80 ꢁC un-
til required. For determination of the inhibition con-
stant (IC50), assays were run as for PBBS binding but
using [3H]flumazenil (2 nM) and cortex membranes at
a final concentration of 250 lg/mL. Samples were incu-
bated for 45 min at 25 ꢁC. Non-specific binding was
determined using flumazenil (20 lM), a selective CBR
ligand.
5.25. [123I]N,N-Diethyl-[5-chloro-2-(4-iodophenyl)indol-3-
yl]glyoxylamide
To a solution of stannane, 5 (125 lg in ethanol (50 lL)
and acetic acid (200 lL)) were added Na123I (311–544
MBq) and per-acetic acid (30%, 25 lL). After 5 min at
room temperature, the reaction was quenched with
Na2S2O5 (51 mg/mL, 100 lL) and NaHCO3 (48–
51 mg/mL, 100 lL). Mobile phase (acetonitrile/0.01 M
ammonium acetate, 3:2 v/v, 350 lL) was added and
the solution was injected onto a semi-preparative C-18
RP HPLC column. The retention time at a flow rate
of 3 mL/min was 16 min. The radiochemical purity
was >96% after formulation in saline.
5.26. Lipophilicity measurements
Samples were analysed using a C18 column (X-Terra,
5l, 4.6 · 250 mm) and a mobile phase of methanol
and 0.1 M phosphate buffer (65:35 v/v, pH 7.5) with a
flow rate of 1 mL/min.24 The log P values were estimat-
ed by comparing HPLC retention times (RT) of test
compounds with retention times of standards having
known log P values. The standards used were aniline,
benzene, bromobenzene, ethylbenzene and trimethyl-
benzene. A calibration curve of log P versus ln RT
was generated. The equation was linear with an r2 of
0.9969. Calculated log P values were found using Chem
Draw Ultra.
Acknowledgments
The authors thank the Australian Institute of Nuclear
Science and Engineering for partial support of the above
work. We also thank Tim Jackson for conducting
lipophilicity measurements.
References and notes
1. Anholt, R. R. H.; De Souza, E. B.; Oster-Granite, M. L.;
Snyder, S. H. J. Pharmacol. Exp. Ther. 1985, 233, 517.
2. De Souza, E. B.; Anholt, R. R. H.; Murphy, K. M. M.;
Snyder, S. H.; Kuhar, M. J. Endocrinology. 1985, 116, 567.
3. Anholt, R. R. H.; Murphy, K. M.; Mack, G. E.; Snyder,
S. H. J. Neurosci. 1984, 4, 593.
4. Cagnin, A.; Brooks, D. J.; Kennedy, A. M.; Gunn, R. N.;
Myers, R.; Turkheimer, F. E.; Jones, T.; Banati, R. B.
Lancet 2001, 358, 461.
5. Banati, R. B.; Newcombe, J.; Gunn, R. N.; Cagnin, A.;
Turkheimer, F.; Heppner, F.; Price, G.; Wegner, F.;
Giovannoni, G.; Miller, D. H.; Perkin, G. D.; Smith, T.;
Hewson, A. K.; Bydder, G.; Kreutzberg, G. W.; Jones, T.;
Cuzner, M. L.; Myers, R. Brain 2000, 123, 2321.
6. Hardwick, M.; Fertikh, D.; Culty, M.; Li, H.; Vidik, B.;
Papadopoulos, V. Cancer Res. 1999, 59, 831.
7. Batra, S.; Alenfall, J. Life Sci. 1992, 51, 1211.
8. Black, K. L.; Ikezaki, K.; Toga, A. W. J. Neurosurg. 1989,
71, 113.
5.27. In vitro binding studies
5.27.1. Peripheral benzodiazepine binding sites. Mito-
chondrial membranes were prepared from kidney ex-
tracts sourced from male Sprague–Dawley rats. The
kidneys were removed, rinsed with ice-cold 50 mM
Tris–HCl buffer (pH 7.4), weighed and finely cut. After
the addition of 20 volumes of ice-cold buffer, the prep-
aration was homogenised and the suspension was cen-
trifuged at 49,000g for 15 min at 4 ꢁC. The pellets were
resuspended in buffer to achieve a protein concentra-
tion of 4 mg/mL. Membranes were stored at ꢀ80 ꢁC
until required.25,26 Protein content in the membrane
suspensions was measured according to a colorimetric
method.27 For determination of the inhibition constant
(IC50), assays were run in triplicate with concentrations
of test compounds ranging from 10ꢀ5 to 10ꢀ10 M in

3946
T. P. Homes et al. / Bioorg. Med. Chem. 14 (2006) 3938–3946
9. Black, K. L.; Ikezaki, K.; Santori, E.; Becker, D. P.;
Vinters, H. V. Cancer 1990, 65, 93.
10. Dumont, F.; De Vos, F.; Versijpt, J.; Jansen, H. M.; Korf,
J.; Dierckx, R. A.; Slegers, G. Eur. J. Nucl. Med. 1999, 26,
194.
18. Kozikowski, A. P.; Ma, D.; Brewer, J.; Sun, S.; Costa, E.;
Romeo, E.; Guidotti, A. J. Med. Chem. 1993, 36, 2908.
19. Gildersleeve, D. L.; Van Dort, M. E.; Johnson, J. W.;
Sherman, P. S.; Wieland, D. M. Nucl. Med. Biol. 1996,
23, 23.
11. Cagnin, A.; Myers, R.; Gunn, R. N.; Lawrence, A. D.;
Stevens, T.; Kreutzberg, G. W.; Jones, T.; Banati, R. B.
Brain 2001, 124, 2014.
12. Mattner, F.; Katsifis, A.; Staykova, M.; Ballantyne, P.;
Willenborg, D. O. Eur. J. Nucl. Med. Mol. Imag. 2005, 32,
557.
20. Katsifis, A.; Barlin, G.; Mattner, F.; Dikic, B. Radiochim.
Acta 2004, 92, 305.
21. Primofiore, G.; Da Settimo, F.; Taliani, S.; Simorini, F.;
Patrizi, M. P.; Novellino, E.; Greco, G.; Abignente, E.;
Costa, B.; Chelli, B.; Martini, C. J. Med. Chem. 2004, 47,
1852.
13. Charbonneau, P.; Syrota, A.; Crouzel, C.; Valois, J. M.;
Prenant, C.; Crouzel, M. Circulation 1986, 73, 476.
14. Versijpt, J. J.; Dumont, F.; Van Laere, K. J.; Decoo, D.;
Santens, P.; Audenaert, K.; Achten, E.; Slegers, G.;
Dierckx, R. A.; Korf, J. Eur. Neurol. 2003, 50, 39.
15. Davies, L. P.; Barlin, G. B.; Selley, M. L. Life Sci. 1995,
57, 381.
22. Al-Azawe, S.; Sarkis, G. Y. J. Chem. Engin. Data 1973, 18,
109.
23. Calvaire, A.; Pallaud, R. Ecole Natl. Super. Chim. 1960,
250, 3194.
24. Dumont, F.; Waterhouse, R. N.; Montoya, J. A.; Matt-
ner, F.; Katsifis, A.; Kegeles, L. S.; Laruelle, M. Nucl.
Med. Biol. 2003, 30, 435.
16. Katsifis, A.; Mattner, F.; Dikic, B.; Papazian, V. Radio-
chim. Acta 2000, 88, 229.
17. Romeo, E.; Auta, J.; Kozikowski, A. P.; Ma, D.;
Papadopoulos, V.; Puia, G.; Costa, E.; Guidotti, A.
J. Pharmacol. Exp. Ther. 1992, 262, 971.
25. Fonia, O.; Weizman, R.; Zisman, E.; Ashkenazi, R.;
Gavish, M. Eur. J. Pharmacol. 1995, 293, 335.
26. Awad, M.; Gavish, M. J. Neurochem. 1987, 49, 1407.
27. Lowry, O. H.; Rosenburg, N. J.; Farr, A. L.; Randall, R.
J. J. Biol. Chem. 1951, 193, 265.