New thalidomide analogues derived through Sonogashira or Suzuki reactions and their TNF expression inhibition profiles

Article abstract of DOI:10.1016/j.bmc.2009.12.001

A library of new thalidomide C4/5 analogues containing either a phenyl or alkyne tether were synthesized using Sonogashira or Suzuki cross coupling reactions from their aryl halogenated precursors. All thalidomide analogues were tested for their ability to inhibit the expression of the proinflammatory cytokine Tumor Necrosis Factor (TNF). More explicitly the use of a novel reporter system utilizing the promoter region of the TNF gene in a human T-cell line provided a rapid and effective measure of NFκB transcriptional activity. Several compounds either containing either an aryl-isobutyl or aryl-isopropoxy group were the most effective in inhibiting TNF expression, and were several times more active than thalidomide itself. Five of the more active derivatives indicated an apoptotic response while one of these compounds, containing an aldehyde tether, showed possible influence of cell cycling effects.

Full text of DOI:10.1016/j.bmc.2009.12.001

Bioorganic & Medicinal Chemistry 18 (2010) 650–662
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New thalidomide analogues derived through Sonogashira or Suzuki
reactions and their TNF expression inhibition profiles
*
Scott G. Stewart , Carlos J. Braun, Sze-Ling Ng, Marta E. Polomska, Mahdad Karimi, Lawrence J. Abraham
The School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia Crawley, WA 6009, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
A library of new thalidomide C4/5 analogues containing either a phenyl or alkyne tether were synthe-
sized using Sonogashira or Suzuki cross coupling reactions from their aryl halogenated precursors. All
thalidomide analogues were tested for their ability to inhibit the expression of the proinflammatory cyto-
kine Tumor Necrosis Factor (TNF). More explicitly the use of a novel reporter system utilizing the pro-
Received 26 September 2009
Revised 28 November 2009
Accepted 2 December 2009
Available online 6 December 2009
moter region of the TNF gene in a human T-cell line provided a rapid and effective measure of NFjB
transcriptional activity. Several compounds either containing either an aryl-isobutyl or aryl-isopropoxy
group were the most effective in inhibiting TNF expression, and were several times more active than tha-
lidomide itself. Five of the more active derivatives indicated an apoptotic response while one of these
compounds, containing an aldehyde tether, showed possible influence of cell cycling effects.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Thalidomide
Tumour necrosis factor (TNF)
Suzuki reaction
Sonogashira reaction
TNF expression inhibition
O
O
3'
H
1'
1. Introduction
1
3
N
N
O
The racemic drug thalidomide, [(R,S)-2-(2,6-dioxo-3-piperidi-
nyl)-1H-isoindole-1,3(2H)-dione (1) (Fig. 1)] tragically was admin-
istered to pregnant woman in the 1950’s as a treatment for
insomnia and as an antiemetic agent. This market release, prior
to a full understanding of the pharmacological profile of both enan-
tiomers, was later found to be premature as the C3’ R-stereoisomer,
(R)-1 was responsible for a sedative response and the S-isomer (S)-
1 had teratogenic properties.1–4 As a result, in 1962 this popular
commercial drug was withdrawn, although not before 10,000 in-
fants with various limb malformations were born.
5
H
5'
4
O
1
Figure 1. Thalidomide 1.
(1) to treat the as-yet-incurable form of bone marrow cancer, mul-
tiple myeloma.3
At a cellular level thalidomide (1) influences several processes
including peptidase inhibition, glucosidase inhibition, androgen
receptor antagonism and (cyclooxygenase) COX inhibition.13 This
assortment of cellular processes indicates that thalidomide (1)
may have many distinct downstream cellular targets. However,
the precise molecular mechanism of action of thalidomide (1)
has been difficult to determine primarily due to its differing
in vitro versus in vivo activities. These discrepancies are thought
to result from the formation of up to 100 in vivo metabolic break-
down products.14 One of the most studied biological activities
influenced by thalidomide (1), is the inhibition of the pro-inflam-
matory cytokine, tumour necrosis factor (TNF) expression.3,15 TNF
is a central regulator of the inflammatory cascade controlling many
effector pathways, the main ones being anti-angiogenic, anti-
inflammatory and immuno-modulatory. At a molecular level the
mode of action of thalidomide (1) in suppressing TNF expression
is thought to involve the inhibition of the nuclear factor kappa-
Currently, with the knowledge of the previous side effects, there
has been a resurgence in the use of thalidomide as a pharmaceuti-
cal agent. In 1998, Celgene received FDA approval to use thalido-
mide (1), (Thalomid™), for the treatment of erythema nodosum
leprosum (ENL) based on results from earlier serendipitous treat-
ment of leprosy patients in 1962.5,6 The efficacy of thalidomide
(1) in the treatment of various proinflammatory and autoimmune
conditions has lead to greater interest in therapeutic applications.
At present, this reborn drug is being evaluated in the treatment of
many disease states such as rheumatoid arthritis,7,8 Behçet’s dis-
ease,8 Crohn’s disease,9 HIV/AIDS related illnesses,9 mesothle-
oma,10 tuberculosis11 and sarcoidosis.12 However, one of the
most promising avenues of investigation is the use of thalidomide
* Corresponding author. Tel.: +61 8 6488 3180; fax: +61 8 6488 1005.
E-mail address: sgs@cyllene.uwa.edu.au (S.G. Stewart).
light-chain-enhancer of activated
B cells (NFjB) signalling
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.001

S. G. Stewart et al. / Bioorg. Med. Chem. 18 (2010) 650–662
651
pathway16 and more specifically by inhibiting the activity of the
B kinase, IKK
17 As part of our ongoing studies into the relation-
ship between thalidomide (1) and TNF expression, we decided to
marked improvements in TNF expression inhibition by these C4
and C5 substituted derivatives synthesized within our group and
the previous illustrated cases, we sought to investigate this region
of thalidomide further. We also looked to exploit the synthetic util-
ity of our halothalidomides 10 and 11 (Scheme 1) with other cross
coupling reactions at these two carbon centres. Our focus turned to
Ij
a.
screen new analogues for enhanced ability to specifically inhibit
the NFj
B pathway.18
With the growing attention this compound has received, it is
expected that the number of thalidomide analogues will grow in
the coming years. Currently in the literature several classes of tha-
lidomide derivatives have been described. Thalidomide analogues
O
O
which greatly improve water solubility, for example CC305219
2
NH
and the valine ammonium salt derivative 3 (Fig. 2) have an im-
proved ability to inhibit TNF.20 Structural modifications to the left
hand phthalimide ring system have also seen great improvements
in activity, for example the two marketed drugs lenalidomide
(Revlimid™) 4 and Actimid™, 5,6,21 containing amino groups at
C4, have shown excellent activity in several assays including TNF
expression inhibition. As such, these two compounds, have been
marketed as immunomodulatory drugs (iMiD’s).3 The C5-hydro-
xy-thalidomide 6, listed as a potential metabolite, caused a loss
of TNF inhibiting activity when compared to the inhibition level
of thalidomide (1) itself.22 In other biological evaluations the C5-
carboxylic acid analogue 7 displayed improvements in basic fibro-
blast growth factor (bFGF) inhibition along with an improved
water solubility.
In view of the fact that these highlighted simple analogues func-
tionalised in the C4 and C5 position have been shown to improve
the inhibitory activity of TNF expression, we have focussed atten-
tion on preparing more complex analogues which could enhance
potential protein interactions and in turn inhibitory activity.
These requirements led us to investigate the production of new
thalidomide olefinic analogues via a Heck cross coupling reac-
tion.18 The bioactivity of each analogue was determined using a
TNF inhibition assay.18,23 From this initial study two key analogues,
the furan derivative 8 and the vinyl butoxy derivative 9 (Fig. 2)
showed a marked improvement in repression of TNF expression.
Unfortunately, in the analysis of these compounds an accurate
structure–activity relationship (SAR) was difficult to achieve due
to the complexity of the attached groups at C4 and C5. Syntheti-
cally, the Heck cross coupling procedure allowed for ease of ana-
logue production in good to high yields (ca. 55–90%) depending
on the electronic nature of the coupling partner. Considering these
N
O
O
12
Y
= Spacer Group
Y
= Functional Group
O
O
O
O
NH
O
NH
N
N
O
O
O
14
13
R
R
Suzuki
Reaction
Sonogashir a
Reaction
O
O
H
N 1'
1
3
3'
N
O
5
H
X
O
10 X = I (C4) or 11 X = Br (C5)
Scheme 1. Introduction of phenyl and alkyne motifs to the thalidomide backbone
via Sonogashira and Suzuki cross coupling reactions.
OMe
O
O
OMe
OMe
O
O
O
O
NH2.HCl
O
O
H
N
N
1'
N
N
3'
N
N
N
O
O
5
3
4
O
O
O
O
NH2
2
3
4
O
O
O
H
O
H
O
H
N
3'
3'
3'
N
O
N
O
N
5
5
HO
HO2C
4
O
O
NH2
5
7
6
O
O
O
N
H
O
H
N
N
O
N
O
4
4
O
O
BuO
8
O
9
Figure 2. Thalidomide analogues in the literature and produced by the Stewart group.

652
S. G. Stewart et al. / Bioorg. Med. Chem. 18 (2010) 650–662
two of the most reliable and chemoselective cross coupling reac-
tions, the Sonogashira and Suzuki transformations.24,25 Such cross
coupling methodologies are frequently used in large scales in
chemical industry and in new medicinal chemistry research
programs.26,27
latter compound with acetic anhydride gave the desired cyclised
iodoanhydride 17 in 68% yield. The second left hand ring fragment,
5-bromophthalic anhydride was purchased from Wako chemi-
cals.31 Condensation of either iodoanhydride 17 or bromophthalic
anhydride with the salt of aminoglutaramide 15 produced the re-
quired halogenated cross coupling partners 10 and 11.
2. Results and discussion
2.1. Synthetic approaches
3. Cross-coupling reactions
To optimise the conditions of Suzuki reaction for the brominated
thalidomide 11, seven sets of catalytic conditions using the 4-acetly-
phenyl boronic acid 20 or the 4-isobutyl boronic acid 21 were at-
tempted (Table 1). Reasonable results were achieved using
Pd(dppf)Cl2 or the more traditional Suzuki cross coupling coupling
catalysts, that is, Pd(PPh3)4. However, under a Pd2(dba)3, [(tBu)3-
PH]BF4 and two base catalytic system (NaOH and dicyclohexyl-N-
methyl amine) the cross coupling was found to be the most efficient
in producing compounds 22 and 23.32 The two catalytic systems,
(Table 1, entries 5 and 8) were clearly the most suited to the thalid-
omide scaffold and provided a range of thalidomide analogues in
mostly in good to excellent yields. The only exception in these cases
was the coupling of the p-phenol boronic acid which proceeded in
only 51%, possibly due to the solubility and reactivity of the phenox-
ide ion. In these initial sets of conditions no homo coupling product
was observed and thus exploration of the boronic ester equivalent
reagents was not required.
In this investigation we set out to identify reliable structural
improvements to the thalidomide skeleton and in the process con-
struct a SAR for the inhibition of TNF expression. In further probing
the region of space around C4 and C5 and taking our initial active
analogues into account, we initially looked at a system which con-
tained a hydrocarbon spacer unit and a heteroatom containing
functional group (i.e., generic derivative 12). Initially we conceived
that a para-aryl substitution pattern meets this prerequisite. Syn-
thetically, the Suzuki cross coupling has been proven to efficiently
introduce new aryl groups through transmetallation of boronic
acids and esters.25 In this study we also envisaged an alkyne spacer
to also explore this region of space (Scheme 1). This fragment could
also be introduced through a second cross coupling reaction, the
Sonogashira reaction (a modified Castro–Stevens reaction).24
The piperidine-2,6-dione ring fragment of the thalidomide core
was prepared by first synthesising the trifluoroacetic acid salt of
aminoglutaramide 15 (Scheme 2). Thus, following the procedure
of Muller or Brown, the commercially available tert-butoxycar-
Following the clean conversion of the Suzuki reaction we fo-
cused our attention to installing an alkyne through the Sonogashira
reaction. This Pd(PPh3)2Cl2 mediated cross coupling proceeded in
good yields (48–78%) for most reactions where an electron donat-
bonyl-L-glutamide 16 was cyclised through a condensation reac-
tion and subsequently deprotected to afford compound 15 in
good yields ca. 72%.28 The first halogented left hand ring fragment
of thalidomide, iodinated phthalic anhydride 17, was prepared
from 2,3-dimethylaniline (18). Accordingly, an initial Sandmeyer
type iodination to the corresponding dimethyl iodobenzene
(49%)29 followed by a potassium permanganate mediated dibenzy-
lic oxidation furnished the diacid 19 (41%).30 Treatment of this
ing group in the
a-position of the alkyne was used (Scheme 3).
These isolated yields agree with the literature findings where high
yields are normally observed with terminal alkynes of similar elec-
tronics and not with substrates containing an electron withdraw-
ing group. Unfortunately, we also required a substrate with an
electron withdrawing substituent in this position for SAR investi-
O
H
O
N
NH
Boc
OH
a
b
TFA.H2N
O
16
O
15
NH2
vi
O
OH
c
e
O
O
O
OH
d
f
NH2
18
I
O
I
19
17
g
O
N
O
O
O
O
NH
NH
O
O
N
Br
O
I
10
11
Scheme 2. Reagents and conditions: (a) CDI, DMAP, THF, reflux, 24 h, 76%; (b) 1:1 TFA/CH2Cl2, 22 °C, 1 h, 95%; (c) H2O, HCl (concd), NaNO2, ꢀ15 °C, KI (aq), then 55 °C, 5 min
then 16 h, rt, 66%; (d) H2O, KMnO4, 80 °C, 4 days, 41%; (e) Ac2O, reflux, 3 h, 68%; (f) NEt3, 15, 5-bromophthalic anhydride, THF, reflux, 48 h, 63%; (g) NEt3, 15, 17, THF, reflux,
24 h, 56%. CDI: 1,1-carbonyldiimidazole; DMAP: 4-dimethylaminopyridine; TFA: trifluoroacetic acid.

S. G. Stewart et al. / Bioorg. Med. Chem. 18 (2010) 650–662
653
Table 1
Optimisation of Suzuki reaction between 5-bromothalidomide 11 and boronic acids 20 and 21
O
O
O
H
N
Pd0 conditions
3'
N
O
B(OH2
)
4
20
O
O
O
H
22
O
N
O
3'
N
O
Br
4
O
H
B(OH2
)
O
N
11
21
3'
N
O
Pd 0 conditions
4
O
23
Entry
Product and boronic acid
Catalyst (mol %)
Phosphine (mol %)
Base(s)
Yield (%)
1
2
3
4
5
6
7
8
22 and 20
22 and 20
22 and 20
22 and 20
22 and 20
23 and 21
23 and 21
23 and 21
Pd(PPh3)4, 10 mol %
Pd2(dba)3,c 5 mol %
Pd2(dba)3,c 5 mol %
Pd(dppf)Cl2,a 10 mol %
Pd2(dba)3,c 5 mol%
Pd(OAc)2, 10 mol %
Pd2(dba)3, 10 mol %
Pd(dppf)Cl2,a 10 mol %
CsF2
CsF2
17
33
70
54
72
19
9
(t-Bu)3PHBF4, 10 mol %
(t-Bu)3PHBF4, 10 mol %
NaOH, Cy2NMe
NaOH, Cy2NMeb
(t-Bu)3PHBF4, 10 mol %
dppf, 20 mol %
dppf, 20 mol %
NaOH
NaOH
NaOH, Cy2NMeb
90
a
b
c
dppf = 1,10-Bis(diphenylphosphino)ferrocene.
Cy2NMe = Dicyclohexyl-N-methyl amine.
Pd2(dba)3 = Tris(dibenzylideneacetone)dipalladium(0).
O
O
H
O
O
Sonogashira
Cross Coupling
N
NH
N
O
N
O
(a)
H
Br
O
O
11
24
Sonogashira
Cross Coupling
(b)
O
O
O
O
O
NH
(c)
NH
N
O
N
O
O
O
OH
26
25
Scheme 3. Reagents and Conditions: (a) Pd(PPh3)2Cl2 (10 mol %), CuI (10 mol %), iPrNEt2, THF, reflux, 18 h, 76%; (b) Pd(PPh3)2Cl2 (10 mol %), CuI (10 mol %), iPrNEt2, THF,
reflux, 4 h, 48%; (c) Dess–Martin periodinane, CH2Cl2, rt, 1 h, 76%.
gations. Thus, the respective coupled alcohol 25 was treated with
Dess–Martin periodinane to yield the required aldehyde 26 in
76% yield.
to determine the effects on the NF
sure of immunomodulatory or anti-inflammatory activity. To effec-
tively measure inhibition of NF B pathway signalling by each
jB activation pathway, as a mea-
j
Once again only small differences between the cross couplings
of the iodo and bromo-thalidomide yields (10 and 11) could be ob-
served. In the case of the Sonogashira reactions the C4 position
seems to be more reactive, possibly due to the enhanced oxidative
addition normally observed with aryl iodide substrates. Neverthe-
less, in the context of creating methodology which can be used effi-
ciently to generate a variety of complex analogues for biological
screening these two described methods seem to be highly
chemoselective.
analogue, a TNF transcriptional reporter cell line was used. The
green fluorescent protein (GFP) reporter gene, under the control
of the NFjB-responsive human TNF promoter, was inserted into
the genome of the human T cell line, Jurkat to generate the reporter
line, FRT-Jurkat TNF, as previously described.23,35 As a measure of
TNF promoter activity, GFP activity was quantitated by flow
cytometry. This method has the added advantage of being able to
easily assess the cellular toxicity of each compound, (by comparing
forward- and side-scatter as a measure of cellular size and granu-
larity) during flow cytometry. The cell population in each assay
that exhibited low granularity were considered to be dead. This
was confirmed by staining with propidium iodide, a fluorescent,
DNA-intercalating agent. All thalidomide derivatives were assayed
4. TNF expression inhibition studies
While many studies involving the inhibition of TNF production
have been reported22,33,34 we have developed a more specific assay
in triplicate at concentrations of 100 lM and the percentage inhi-

654
S. G. Stewart et al. / Bioorg. Med. Chem. 18 (2010) 650–662
Table 2
bition of TNF expression (relative to TNF expression from solvent
treated control cells) for each compound measured. As our previ-
ous study showed significant solvent effects at 1% dimethylsulfox-
ide (DMSO), the final concentration in the current study was
lowered to 0.1%. At this concentration, the solvent has very little ef-
fect on TNF reporter gene expression.
The Suzuki derived aryl based analogues were tested for their
ability to inhibit TNF expression and their significance was mea-
sured verses the thalidomide (1) control (Table 2). Each of the
derivatives was selected based on possible receptor interactions,
although several derivatives were assumed to have increased
water solubility, possibly also enhancing the TNF expression inhi-
bition. Initially derivatives (27 and 34) containing a phenyl substi-
TNF expression inhibition of new aryl based thalidomide derivatives
O
O
H
1'
1
N
3'
N
O
5
H
5'
3
4
O
R
Compounda,b
R (carbon substitution)
H
% Inhibition
p Valuec
Thalidomide 1
6%
—
27
28
14
0.0324
(C4)
(C4)
tuent investigated the likelihood of
p
-stacking or hydrophobic
10
86
0.2762
interactions in potential binding pockets. Each of these derivatives
improved the TNF repressive activity of thalidomide by approxi-
mately twofold, however this result fell just outside the (p <0.05)
confidence interval. Encouraged by this slight improvement we de-
vised a series of derivatives containing a p-substituent considered
to be a linear extension away from the thalidomide core. While the
two toluyl derivatives (28 and 35) showed no significant improve-
ment, the isobutyl derivatives (23 and 29) exploring possible inter-
actions with a hydrophobic binding pocket showed an exceptional
improvement of TNF expression inhibition. Accompanying this
activity for compounds 23 and 29 was high cell death. However,
dead cells were excluded and only viable cells were assayed for
TNF repressive activity indicating that the two activities were sep-
arate and distinct. Of the other aryl derivatives examined ethers 32
and 38, isosters of compounds 23 and 29, also displayed good TNF
inhibitory activity suggesting the shape of the para group maybe
important. Analogue 38 in particular had a good cell viability count
compared with each of the previously discussed derivatives.
An alternative explanation to altered receptor interactions for
the increased activities, may be the polarity and/or water solubility
of the indicated compounds. Many of the derivatives have similar
inhibition values regardless of whether the functional group tether
is located at position C4 or C5. Similar observations have been re-
ported in the literature.19,36
29
0.0001
(C4)
(C4)
OH
O
30
31
19
14
0.0092
0.0615
NH2
(C4)
(C4)
O
32
33
47
17
0.0009
0.0425
O
(C4)
34
35
12
14
0.0926
0.0503
(C5)
(C5)
23
90
0.0001
(C5)
The TNF inhibition level was also determined for the alkyne
based analogues (Table 3). Compounds that showed significant
improvement were the two tBu derivatives (39 and 24), surpris-
ingly also containing non-polar fragments like the previously dis-
cussed aryl butyl compounds (29 and 23). Both of these
derivatives exhibit approximately a fourfold increase in TNF inhibi-
tion activity when compared to thalidomide (1). Alcohol derivative
45 also displayed significantly increased activity compared to tha-
lidomide (1). Perhaps the most interesting derivative was com-
pound 26, conceived to test the hypothesis that an electron
withdrawing group attached to the alkyne may improve the inhibi-
tion of TNF expression. This compound displayed a percent inhibi-
tion of 85% in comparison to the thalidomide control at 5%.
The derivatives which displayed a significantly higher TNF
activity in the initial study (23, 26, 29, 32 and 38) were further
OH
36
37
11
10
0.1639
0.0557
(C5)
O
NH2
O
(C5)
(C5)
O
38
22
59
26
0.0047
0.0084
(C5)
a
Percentage inhibition was measured as the difference between GFP expression
in the presence of 0.1% solvent alone, and each compound at 100 lM. The assay was
carried out using the FRT-Jurkat TNF reporter cell line.
All compounds were assayed in triplicate and as a racemic mixture.
The p values which have a significant difference compared to thalidomide 1 (p
<0.05) are italicised. Significance was estimated using the unpaired Student’s t-test.
b
c
examined at one tenth the initial concentration (10 lM), to deter-
mine if there was retention of activity in comparison to thalido-
mide (1) (Table 4). Each of these compounds were also
examined for their influence on cell viability. As was expected
compounds 23, 29, 32 and 38 showed a dramatic decrease in
TNF inhibition, however at this concentration compound 38 was
still threefold more active than thalidomide 1 with a good cell via-
bility count. In contrast, compound 26 also indicated a high TNF
5. Characterisation of the cytotoxicity of compounds 23, 26, 29,
32 and 38
Given the high degree of cell death elicited by compounds 23,
inhibition at 10
(1), however the rate of cell death remained high. Decreasing
the concentration to 1 M greatly improved the viable cell count.
However activity was also reduced almost to the level of thalido-
mide (1).
l
M, approximately 18 times that of thalidomide
26, 29, 32 and 38 at 100 lM, we were interested in determining
whether the cellular response was apoptotic in nature as this cell
death pathway has been successfully targeted in a number of
cancers to selectively kill the malignant cell.37,38 As thalidomide
has been shown to be effective in the treatment of a number of
l

S. G. Stewart et al. / Bioorg. Med. Chem. 18 (2010) 650–662
655
Table 3
Table 4
TNF expression inhibition of new alkyne based thalidomide derivatives
TNF expression inhibition and cell viability for lower concentrations of compounds
23, 26, 29, 32 and 38
O
O
H
1
Compound
Concentration
M)
TNF inhibitiona,b
(%)
Viable cellsc
(%)
N
1'
3'
(
l
N
O
5
Thalidomide (1)
10
10
10
10
10
10
1
4
5
7
H
5'
3
4
23
29
32
38
26
26
26
89
90
90
90
25
95
96
O
R
Compound
R (carbon substitution)
% Inhibitiona,b
5
p Valuec
8
10
86
5
Thalidomide 1
H
—
39
40
24
13
0.0383
0.1
4
(C4)
(C4)
a
Percentage inhibition was measured as the difference between GFP expression
in the presence of 0.1% solvent alone, and each compound at the indicated con-
centration. The assay was carried out using the FRT-Jurkat TNF reporter cell line.
All compounds were assayed in triplicate and as a racemic mixture.
Viable cells were counted as being indistinguishable from solvent treated cells
with respect to forward and side scatter characteristics, using the flow cytometer
(see gate in Fig. 3).
0.6821
b
c
OH
CN
41
42
43
6
9
7
0.6433
0.2230
0.3103
(C4)
(C4)
(C4)
(C5)
Table 5
OH
Compounds 23, 26, 29, 32, 38 and apoptosis
Compound
Concentration
Cell deatha,b
Apoptosisc (%)
24
44
45
19
6
0.0019
0.0553
0.0287
Untreated
DMSO
Thalidomide
Thalidomide
—
0.10%
100
7
7
7
6
21
6
94
7
33
8
51
14
86
7
8
8
9
7
22
7
96
7
30
8
34
12
84
7
l
M
M
(C5)
10
l
23
23
29
29
32
32
38
38
26
26
26
100
lM
10
l
M
OH
20
100
lM
(C5)
(C5)
10
l
M
100
lM
46
47
25
26
10
12
10
85
0.2153
0.2130
0.0344
0.0002
HN
10
l
M
100
lM
CN
10
10
l
l
M
M
(C5)
1
lM
0.1
l
M
4
7
OH
O
(C5)
a
The assay was carried out using the Jurkat cell line; cell death was measured as
the percentage of cells showing a decrease in cell size and granularity (forward and
side scatter) in flow cytometric analysis.
All compounds were assayed in triplicate and as a racemic mixture.
Apoptosis was measured flow cytometrically as the percentage of cells that
showed an increase in green fluorescence following staining for cleaved caspase 3.
d
(C5) d
b
a
Percentage inhibition was measured as the difference between GFP expression
in the presence of 0.1% solvent alone, and each compound at 100 lM. The assay was
carried out using the FRT-Jurkat TNF reporter cell line.
c
b
All compounds were assayed in triplicate and as a racemic mixture.
The p values which have a significant difference compared to thalidomide 1 (p
c
<0.05) are italicised. Significance was estimated using the unpaired Student’s t-test.
Indicated compound was produced through Dess–Martin oxidation of the cor-
responding alcohol 25.
d
tible to this derivative’s mode of action. The weakly positive popu-
lation has entered the cell cycle later than the strongly positive
population. These results further support the notion that com-
pound 26 may be more active on cells undergoing vigorous cell
division, such as malignant cancer cells, than generally quiescent
normal tissue.
To investigate whether compound 26 was apoptotic when used
to treat normal cells rather than a transformed leukemic cell such
as Jurkat, peripheral blood mononuclear cells (PBMCs) were iso-
lated from healthy volunteers, activated and treated with com-
pound 26 or thalidomide (1). The effect on expression of the
endogenous TNF was measured by quantitative RT-PCR (Fig. 3c).
cancers, it is possible that the cytotoxicity may be used to advan-
tage in therapeutic applications. Each of the compounds were
used to treat Jurkat T cells at the concentrations shown in Table
5. The degree of apoptosis was measured and compared with cell
death measured by cellular size and granularity. The results indi-
cate that for each of the compounds, all of the cells that were
undergoing cell death were in fact apoptotic, indicating that these
compounds had both TNF inhibitory activity and also apoptotic
activity.
The results show that compound 26 at 10
TNF expression and even at 1 M, significant activity was evident
and showed inhibition similar to the level for thalidomide at
100 M. None of the treatments resulted in significant cell death
lM strongly repressed
l
Inspection of the apoptosis flow cytometry histograms for these
compounds revealed that compound 26 showed unusual addi-
l
tional characteristics ( Fig. 3). At 1
(94%) were viable with 6% undergoing apoptosis (Fig. 3a). However,
at 10 M, 86% of the cells were apoptotic, but showed two distinct
lM the majority of the cells
compared to the DMSO control.
l
populations, one that was weakly positive and one that was
strongly positive for the apoptosis phenotype (Fig. 3b). This result
is consistent with a cell-cycle specific activity for compound 26,
where only cells at a particular stage of the cell cycle, are suscep-
6. Conclusion
We have described the rapid production of several thalidomide
based derivatives through two cross couplings in reasonable to

656
S. G. Stewart et al. / Bioorg. Med. Chem. 18 (2010) 650–662
Figure 3. Treatment with compound 26 results in apoptosis and inhibition of TNF mRNA expression. (a and b) Flow cytometry of cells treated with either 1 lM (a) or 10 lM
(b) compound 26 for 24 h. Viability is shown by the circled regions in the side scatter (SSC) versus forward scatter (FSC) plot (left panels). Right panels represent histograms of
cell counts following treatment with the fluorescent substrate (FITC) specific for cleaved caspase-3 as a measure of the percentage of cells undergoing apoptosis. One
representative of three independent experiments is shown. Horizontal gates [6%, panel (a) and 86%, panel (b)] represent the percentage of cells considered positive for
apoptosis. (c) Freshly isolated PBMCs were activated and cultured in the presence of DMSO, thalidomide 1 (100 lM or 10 lM) or compound 26 (10 lM or 1 lM) for 2 h. Total
RNA was extracted and assayed using TNF-specific quantitative RT-PCR. The results were normalised against the house-keeping gene b-actin (shown as TNF Relative
*
Expression). = significantly different to DMSO control (P <0.05). Error bars represent standard error of the mean.
excellent yields from their halogenated precursors. Biological re-
sults indicate that several thalidomide derivatives (23, 29, 32 and
38) exhibited significant increases in TNF expression inhibitory
activity compared to thalidomide (1). Compound 38 displayed a
superior TNF inhibitory activity to thalidomide (1) at both 100
of the mode of action of thalidomide. The aldehyde 26, displayed
extraordinary TNF repression as well as cytotoxicity in the form
of apoptosis. However, the apoptotic response to compound 26 ap-
pears to be selective for transformed or actively dividing cells as
PBMCs did not undergo apoptosis following treatment. Compound
26 is being investigated further with respect to its potential selec-
tive apoptotic and cell cycle properties in other malignant cell
types.
and 10 lM. Several of these active derivatives include a short
non-polar chain attached to either an aryl or alkyne spacer group,
however it is not definitive whether the position of the derivatisa-
tion is important for improved TNF expression inhibition or other
factors such as solubility and polarity. These factors are currently
being investigated in separate studies involving the determination
The activities of the new derivatives produced in this study sup-
port the notion that further modification of thalidomide to produce
more active TNF inhibiting analogues for the treatment of disease

S. G. Stewart et al. / Bioorg. Med. Chem. 18 (2010) 650–662
657
states where immunotherapeutic intervention is indicated, may
prove clinically useful.
Method C: Phenylboronic acid (1.0–1.5 equiv), was added to a
suspension of halogenated-thalidomide 10 or 11 (1.0 equiv),
Pd(dppf)Cl2 (10 mol %), NaOH (1.0 equiv), N,N-dicyclohexylmethyl-
amine (1.0 equiv) and THF at room temperature. The mixture was
then stirred at reflux for between 2 and 18 h before being fused to
silica and passed through a short silica plug eluting with ethyl ace-
tate. The crude organic eluent was then fused to silica and sub-
jected to column chromatography (conditions specified). The
resulting product, following concentration of the appropriate frac-
tions, was used directly for biological assay or subsequently recrys-
tallised from ethyl acetate. The yields are specified for each
individual case.
7. Experimental section
7.1. Chemistry
Nuclear Magnetic Resonance (NMR) spectra were recorded on a
Varian INOVA 300 (1H at 300.14 MHz and 13C at 75.47 MHz), Bru-
ker AV 500 (1H at 500.13 MHz and 13C at 125.8 MHz), Varian 400
(1H at 399.86 and 13C at 100.54 MHz) or a Bruker AV 600 (1H at
600.13 MHz and 13C at 150.90 MHz) spectrometer at 25 °C. 1H
and 13C spectra were referenced to the residual (partially) undeu-
terated solvents and are stated in parts per million. Assignments
of 13C resonances were made with the aid of DEPT experiments.
Infrared (IR) spectra were recorded with a PerkinElmer Spectrum
One Spectrometer FT-IR spectrometer. Samples were analysed as
thin films on NaCl discs. Mass Spectra were collected using elec-
tron impact ionisation (EI-MS) on a VG AutoSpec. EI-HRMS was
performed with a resolution of approximately 10,000. All air and/
or moisture sensitive reactions were performed in flame dried
glassware under an argon atmosphere. All solvents were distilled
prior to use, and if used in air and/or moisture sensitive reactions,
were degassed. The degassing procedure was carried out by three
freeze–pump–thaw cycles. Anhydrous solvents were obtained by
distillation from the appropriate drying agent. Thin layer chroma-
tography (TLC) was performed on Merck Silica Gel 60 F254, pre-
coated aluminium sheets. Visualisation of developed plates was
achieved through the use of a 254 nm or 365 nm UV lamp. Column
chromatography was performed using Silica Gel 60 (0.063–
0.200 mm) as supplied by Merck with the eluents indicated. Start-
ing materials and reagents were generally available from Sigma–
Aldrich, Fluka, Merck or Wako. The synthesis of compounds 15,
17, 19 and the trial cross coupling reactions for compounds 22
and 33 are described in the supporting information. Additionally,
1H NMR spectra for compounds 22, 23, 26, 29, 37, 39, 41, 43, 45
and 47 are also illustrated in this section.
7.3. General protocol for the Sonogashira reactions
The alkyne (1.2 equiv) was added in one portion to a suspension
of halogenated-thalidomide 10 or 11 (1.0 equiv), Pd(PPh3)2Cl2
(10 mol %), CuI (10 mol %) and N,N-diisopropylethylamine
(10 equiv) in THF. The ensuing solution was stirred at room tem-
perature (or reflux) for between 4 and 22 h. The resulting mixture
was subsequently fused to silica, filtered through a silica plug elut-
ing with ethyl acetate/hexane. The organic layer was again fused to
silica and purified via flash column chromatography (conditions
specified). The resulting product, following concentration of the
appropriate fractions, was used directly for biological assay or sub-
sequently recrystallised from ethyl acetate. The yields are specified
for each individual case.
7.3.1. (R,S)-2-(2,6-Dioxopiperidin-3-yl)-4-iodoisoindole-1,3-
dione (10)
Triethylamine (3 mL, 21.5 mmol) was added in one portion to a
suspension of 3-iodophthalic anhydride (17) (1.79 g, 6.53 mmol)
and TFA salt 15 (1.61 g, 6.65 mmol) in THF (14 mL) at room tem-
perature. The resulting mixture was heated at reflux for 23 h, be-
fore being cooled to 0 °C. The ensuing precipitate was filtered,
washed with cold THF and dried under reduced pressure to yield
10 as a pale grey solid (1.41 g, 56%). Mp = 304–305 °C; 1H NMR
0
0
(300 MHz, DMSO-d6): d = 1.98–2.12 (m, 1H, H4 H/5 ), 2.52–2.66
0
0
0
0
(m, 2H, H4 /H5 ), 2.82–2.96 (m, 1H, H4 /H5 ), 5.12–5.21 (dd,
0
7.2. General protocol for the Suzuki reaction
J = 12.8 Hz, 5.4 Hz, 1H, H3 ), 7.54–7.62 (m, 1H, H6), 7.93 (dd,
J = 7.4, 0.8 Hz, 1H, Ar-H), 8.27 (dd, J = 7.9, 0.8 Hz, 1H, Ar-H), 11.15
13
0
0
Method A: N,N-Dicyclohexylmethylamine (1.1 equiv) was added
to a suspension of halogenated-thalidomide 10 or 11 (1.0 equiv),
phenylboronic acid/ester (1.0–1.5 equiv), Pd2(dba)3ꢁCHCl3 (5–
10 mol %) and tri-t-butylphosphonium tetrafluoroborate (10–
20 mol %) in THF at ambient temperatures. The resulting mixture
was degassed three times and stirred at reflux for between 2 and
22 h before the solvent was removed under reduced pressure.
The ensuing mixture was subjected to column chromatography
(conditions specified). The resulting product, following concentra-
tion of the appropriate fractions, was used directly for biological
assay or subsequently recrystallised from ethyl acetate. The yields
are specified for each individual case.
Method B: N,N-Dicyclohexylmethylamine (1.1 equiv) is added to
a suspension of halogenated-thalidomide 10 or 11 (1.0 equiv),
phenylboronic acid/ester (1.0–1.5 equiv), Pd2(dba)3ꢁCHCl3 (5–
10 mol %) and tri-t-butylphosphonium tetrafluoroborate (10–
20 mol %), NaOH (1.0 equiv) in THF at ambient temperatures. The
resulting mixture is degassed three times and stirred at reflux for
between 2 and 22 h before the solvent is removed under reduced
pressure. The ensuing mixture was fused to silica and subjected
to column chromatography (conditions specified). The resulting
product, following concentration of the appropriate fractions,
was used directly for biological assay or subsequently recrystal-
lised from ethyl acetate. The yields are specified for each individual
case.
(s, 1H, NH); C NMR (75.4 MHz, DMSO-d6): d = 21.9 (C4 /C5 ),
0
0
0
30.9 (C4 /C5 ), 49.2 (C3 ), 90.5 (C4), 123.3 (Ar-CH), 131.8 (Ar-C),
133.3 (Ar-C), 135.7 (Ar-CH), 145.5 (Ar-CH), 165.5 (C@O), 166.1
~
m
(C@O), 169.8 (C@O), 172.8 (C@O). IR (KBr)
: 3205.0 (N–H),
1726.1 (C@O), 1390.4, 1202.5, 737.9 cmꢀ1; MS EI, m/z (%) = 385
(6) [M+H]Å+, 307 (61), 289 (24), 154 (100), 137 (55). C13H10IN2O4I
(384.1260); EI-HRMS: [M+H]Å+; 384.9685; found [M+H]Å+ 384.9663.
7.3.2. (R,S)-5-Bromo-2-(2,6-dioxopiperidin-3-yl)-isoindole-1,3-
dione (11)
Triethylamine (12 mL, 86.1 mmol) was added to a suspension of
4-bromophthalic anhydride (4.81 g, 21.2 mmol) and TFA salt 15
(5.11 g, 21.1 mmol) in THF (45 mL) at room temperature. The result-
ing mixture was then heated at reflux for 48 h, before being cooled
on ice. The ensuing precipitate was collected, washed with cold
THF and dried under reduced pressure to yield 11 as a pale lavender
solid (4.49 g, 63%). Mp >230 °C; 1H NMR (500 MHz, DMSO-d6):
0
0
0
0
d = 2.01–2.08 (m, 1H, H5 /H4 ), 2.50–2.63 (m, 2H, H5 /H4 ), 2.83–
0
0
0
2.92 (m, 1H, H4 /H5 ), 5.15 (dd, J = 12.9 and 5.4 Hz, 1H, H3 ), 7.85
(dd, J = 8.0 and 0.5 Hz, 1H, H7), 8.08 (dd, J = 8.0 and 1.7 Hz, 1H, H6),
8.14 (dd, J = 1.7 and 0.5 Hz, H4); 13C NMR (125.8 MHz, DMSO-d6):
0
0
0
0
0
d = 21.9 (C4 /C5 ), 30.9 (C4 /C5 ), 49.2 (C3 ), 125.3 (Ar-CH), 126.4 (Ar-
CH), 128.5 (Ar-Br), 130.2 (Ar-C), 133.2 (Ar-C), 137.6 (Ar-CH), 165.9
~
(C@O), 166.4 (C@O), 169.7 (C@O), 172.7 (C@O); IR (KBr)
m
: 3195
(N–H), 1724 (C@O), 1706.7 (C@O), 739.4 cmꢀ1
.

658
S. G. Stewart et al. / Bioorg. Med. Chem. 18 (2010) 650–662
7.3.3. (R,S)-5-(4-Acetylphenyl)-2-(2,6-dioxopiperidin-3-yl)iso-
indoline-1,3-dione (22)
yellow solid (48%); Rf = 0.32 (1:1 ethyl acetate/hexane);
mp = 232–234°C; 1H NMR (300 MHz, DMSO-d6): d = 2.04–2.08
(m, 1H, H4 /H5 ), 2.53–2.63 (m, 2H, H4 /H5 ), 2.85–2.90 (m, 1H, H4 /
0
0
0
0
0
Compound 22 was prepared using the Suzuki general protocol
Method C using 4-acetylphenylboronic acid 20. Flash column chro-
matography (1:1 ethyl acetate/hexane) gave the desired biaryl
compound 22 as a white solid (70%). Rf = 0.23 (1:1 ethyl acetate/
hexane); mp = >230°C; 1H NMR (300 MHz, DMSO-d6): d = 2.07–
0
H5 ), 4.37 (d, J = 6.0 Hz, 2H, CH2), 5.18 (dd, J = 12.9 and 5.4 Hz, 1H,
0
H3 ), 5.47 (t, J = 6.0 Hz, 1H, OH), 7.88–7.94 (m, 3H, Ar-H), 11.14 (s,
13
0
0
1H, NH); C NMR (100.5 MHz, DMSO-d6): d = 22.8 (C4 /C5 , CH2),
0
0
0
31.8 (C4 /C5 , CH2), 50.0 (CH2), 50.4 (C3 , CH), 83.2 (C„C), 95.4
(C„C), 124.8 (Ar-C), 126.5 (Ar-C), 129.7 (Ar-CH), 131.3 (Ar-CH),
132.7 (Ar-CH), 138.4 (Ar-C), 167.3 (C@O), 167.4 (C@O), 170.7
~
(C@O), 173.7 (C@O); IR (KBr,) m: 3444 (N–H), 2210 (C„C), 1716
0
0
0
0
2.11 (m, 1H, H4 /H5 ), 2.50–2.55 (m, 2H, H4 /H5 ), 2.64 (s, 3H, CH3),
0
0
0
2.90 (m, 1H, H4 /H5 ), 5.20 (dd, J = 12.6 and 5.1 Hz, 1H, H3 ), 8.03–
8.11 (m, 5H, Ar-CH), 8.21–8.27 (m, 2H, Ar-CH), 11.10 (s, 1H, NH);
13
C NMR (100.5 MHz, DMSO-d6): d = 22.0 (C4 /C5 ), 26.8 (CH3) 30.9
(C@O), 1384, 1205 (C–O), 1049 cmꢀ1; MS EI, m/z (%): 312 (100)
[M]Å+, 284 (25), 227 (70); EI-HRMS calcd for C16H12N2O5:
312.0746; found: 312.0747.
0
0
0
0
0
(C4 /C5 ), 49.1 (C3 ), 121.8 (Ar-CH), 124.2 (Ar-CH), 127.7 (2 ꢂ Ar-
CH), 129.0 (2 ꢂ Ar-CH), 130.6 (Ar-C), 132.3 (Ar-C), 133.4 (Ar-CH),
136.7 (Ar-C), 142.3 (Ar-C), 145.4 (Ar-C), 166.8 (2 ꢂ C@O), 169.8
~
(C@O), 172.7 (C@O), 197.6 (C@O); IR (KBr)
m
: 3442 (N–H), 1775,
7.3.7. (R,S)-3-(2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-
5-yl)propiolaldehyde (26)
1716 (C@O), 1383, 1268, 1203, 751 cmꢀ1; MS EI, m/z (%): 376
(27) [M]Å+, 261 (98) [MꢀCH3]Å+; EI-HRMS calcd for C21H16N2O5:
376.1059; found: 376.1064.
Dess–Martin periodinane (244 mg, 0.576 mmol) was added in
one portion to
a stirred solution of alcohol 25 (150 mg,
0.480 mmol) in CH2Cl2 (6 mL). The mixture was stirred for 1 h be-
fore being treated with a sodium bicarbonate/sodium thiosulfate
aqueous solution (1:1, 1 mL) in one portion. The reaction mixture
stirred for a further 1 h, transferred to a separatory funnel and ex-
tracted with CH2Cl2 (3 ꢂ 50 mL) and ethyl acetate (2 ꢂ 30 mL). The
combined organic layers were dried (MgSO4) and concentrated un-
der reduced pressure. The resulting solid was subsequently fused
to silica and subjected to flash column chromatography (2:3 ethyl
acetate/hexane) to afford the aldehyde product 26 as a yellow/or-
ange solid (112 mg, 76%); Rf = 0.29 (2:3 ethyl acetate/hexane);
mp = 203–205°C; 1H NMR (400 MHz, DMSO-d6): d = 2.05–2.08
7.3.4. (R,S)-2-(2,6-Dioxopiperidin-3-yl)-5-(4-isobutylphenyl)iso-
indoline-1,3-dione (23)
Compound 23 was prepared using the Suzuki general protocol
Method C using 4-isobutylphenylboronic acid 21. Flash column
chromatography (7:20 ethyl acetate/hexane) gave the title biaryl
compound 23 as a yellow solid (90%); Rf = 0.31 (7:20 ethyl ace-
tate/hexane); mp = 118–120 °C; 1H NMR (300 MHz, DMSO-d6):
d = 0.89 (d, J = 6.6 Hz, 6H, 2 ꢂ CH3), 1.89 (septet, J = 6.9 Hz, 1H,
0
0
CH), 2.06–2.10 (m, 1H, H4 /H5 ), 2.49–2.54 (m, 2H, CH2), 2.58–
0
0
0
0
2.64 (m, 2H, H4 /H5 ), 2.87–2.91 (m, 1H, H4 /H5 ), 5.18 (dd, J = 12.6
0
00
00
0
0
0
0
0
and 5.4 Hz, 1H, H3 ), 7.31 (‘d’, ‘J’ = 8.1 Hz, 2H, H3 /H5 ), 7.76 (‘d’,
(m, 1H, H4 /H5 ), 2.50–2.63 (m, 2H, H4 /H5 ), 2.85–2.92 (m, 1H, H4 /
00
00
0
0
‘J’ = 8.1 Hz, 2H, H2 /H6 ), 7.96 (m, 1H, Ar-CH), 8.17 (m, 2H, Ar-CH),
H5 ), 5.19 (dd, J = 12.8 and 5.2 Hz, 1H, H3 ), 8.03 (d, J = 8 Hz, 1H,
13
0
11.15 (s, 1H, NH); C NMR (100.5 MHz, DMSO-d6): d = 22.0 (C4 /
Ar-CH), 8.04–8.21 (m, 2H, Ar-CH), 9.49 (s, 1H, CHO), 11.15 (s, 1H,
NH); C NMR (100.5 MHz, DMSO-d6) d = 21.8 (C4 /C5 ), 30.8 (C4 /
C5 ), 49.2 (C3 ), 90.0 (C„C), 90.5 (C„C), 124.0, 124.8, 127.2,
131.8, 132.7, 139.2, 165.9 (C@O), 166.2 (C@O), 169.7 (C@O),
13
0
0
0
0
0
0
C5 , CH2), 22.2 (2 ꢂ CH3), 29.6 (CH), 30.9 (C4 /C5 , CH2), 44.2 (CH2),
0
0
0
49.1 (C3 , CH), 121.1 (Ar-CH), 124.0 (Ar-CH), 127.1 (2 ꢂ Ar-CH),
129.5 (Ar-C), 129.8 (2 ꢂ Ar-CH), 132.3 (Ar-C), 132.6 (Ar-CH),
~
m
135.5 (Ar-C), 142.3 (Ar-C), 146.6 (Ar-CH), 166.9 (C@O), 167.0
172.7 (C@O), 178.6 (CHO); IR (KBr)
: 3439 (N–H), 2924, 2194
(C„C), 1722 (C@O), 1391, 1200 cmꢀ1; MS EI, m/z (%): 310 (41)
[M]Å+, 282 (30) [MꢀCHO]Å+, 224 (100); EI-HRMS calcd for
C16H10N2O5: 310.0589; found: 310.0582.
~
m
(C@O), 169.8 (C@O), 172.8 (C@O); IR (KBr)
: 3231 (N–H), 2955,
1778 (C@O), 1716 (C@O), 1383, 1198, 749 cmꢀ1; MS EI, m/z (%):
390 (56) [M]Å+, 348 (26), 347 (100) [MꢀC3H7]Å+, 262 (18), 236
(10); EI-HRMS calcd for C23H22N2O4: 390.1579; found: 390.1575.
7.3.8. (R,S)-2-(2,6-Dioxopiperidin-3-yl)-4-phenylisoindole-1,3-
dione (27)
7.3.5. (R,S)-5-(3,3-Dimethylbut-1-ynyl)-2-(2,6-dioxopiperidin-
3-yl)isoindoline-1,3-dione (24)
Compound 27 was prepared using the Suzuki general protocol
Method A using phenylboronic acid. Column chromatography
(2:3 ethyl acetate/hexane) and subsequent recrystallisation from
ethyl acetate gave the desired 27 as a white solid (66%); Rf = 0.33
(2:3 ethyl acetate/hexane; 1H NMR (400 MHz, CDCl3): d = 2.12–
Compound 24 was prepared using general protocol for the
Sonogashira reaction using 3,3-dimethyl-1-butyne. Flash column
chromatography (7:13 ethyl acetate/hexane) gave the alkyne 24,
as a yellow solid (76%); Rf = 0.25 (7:13 ethyl acetate/hexane);
mp = 222–225 °C; 1H NMR (400 MHz, DMSO-d6): d = 1.32 (s, 9H,
0
0
0
0
2.02 (m, 1H, H4 /H5 ), 2.85–2.60 (m, 3H, H4 /H5 ), 4.94 (dd, J = 12.0,
0
0
0
0
0
3 ꢂ CH3), 2.05–2.08 (m, 1H, H4 /H5 ), 2.50–2.62 (m, 2H, H4 /H5 ),
5.2 Hz, 1H, H3 ), 7.46–7.40 (m, 3H, Ar-H), 7.54–7.48 (m, 2H, Ar-
0
0
0
2.85–2.89 (m, 1H, H4 /H5 ), 5.15 (dd, J = 12.8 and 5.2 Hz, 1H, H3 ),
H), 7.65 (dd, J = 7.5, 1.1 Hz, 1H, H5/H7), 7.84 (dd, J = 7.5, 1.1 Hz,
7.81–7.88 (m, 3H, Ar-H), 11.13 (s, 1H, NH); 13C NMR (100.5 MHz,
1H, H5/H7), 7.74 (dd, J = 7.5, 1.1 Hz, 1H, H6); 13C NMR
0
0
0
0
0
0
DMSO-d6): d = 21.9 (C4 /C5 , CH2), 27.8 (C(CH3)3), 30.4 (3 ꢂ CH3),
(100.5 MHz, DMSO-d6): d = 22.0 (C4 /C5 , CH2), 30.9 (C4 /C5 , CH2),
0
0
0
0
30.9 (C4 /C5 , CH2), 49.1 (C3 ), 77.9 (C„C), 103.5 (C„C), 123.7 (Ar-
CH), 125.5 (Ar-CH), 129.6 (Ar-C), 129.8 (Ar-C), 131.7 (Ar-C), 137.3
(Ar-CH), 166.4 (C@O), 166.6 (C@O), 169.7 (C@O), 172.7 (C@O); IR
~
48.9 (C3 , CH), 122.4 (Ar-CH), 128.0 (Ar-CH), 129.4 (Ar-CH), 135.8
(Ar-CH), 136.4 (Ar-CH), 166.5 (C@O), 166.7 (C@O), 169.8 (C@O),
~
m
172.8 (C@O); IR (NaCl)
: 3247 (N–HN–H), 1699 (C@O), 1393,
(KBr)
m
: 3424 (N–H), 2221 (C„C), 1775, 1725 (C@O), 1385, 1261,
1261, 1199, 1121, 891, 742 cmꢀ1; MS (m/z) = 334 (100) [M]Å+, 249
(37), 248 (38), 224 (40), 222 (21) [MꢀC5H6NO2]+, 180 (27), 152
(51), 151 (24); EI-HRMS calcd for C19H14N2O4: 334.0954; found:
334.0959.
1200 (C–O), 745 cmꢀ1; MS EI, m/z (%): 339 (17) [M+H]Å+, 338
(100) [M]Å+, 184 (42), 169 (33); EI-HRMS: calcd for C19H18N2O4:
338.1267; found: 338.1269.
7.3.6. (R,S)-2-(2,6-Dioxopiperidin-3-yl)-5-(3-hydroxyprop-1-
ynyl)isoindoline-1,3-dione (25)
7.3.9. (R,S)-2-(2,6-Dioxopiperidin-3-yl)-4-p-tolylisoindole-1,3-
dione (28)
Compound 25 was prepared using general protocol for the
Sonogashira reaction using propargyl alcohol. Flash column chro-
matography (1:1 ethyl acetate/hexane) and subsequent recrystalli-
sation from ethyl acetate afforded the desired alkyne 25, as a
Compound 28 was prepared using the Suzuki general protocol
Method A using 4-toluyl pinacolboronic acid ester. Column chro-
matography (2:3 ethyl acetate/hexane) gave 28 as a white solid
(45%). Rf = 0.3 (1:1 ethyl acetate/hexane); Rf = 0.3 (1:1 ethyl ace-

S. G. Stewart et al. / Bioorg. Med. Chem. 18 (2010) 650–662
659
tate/hexane); 1H NMR (400 MHz, DMSO-d6): d = 2.11–2.01 (m, 1H,
4H, Ar-CH), 8.07 (s, 1H, NH2), 11.10 (s, 1H, NH); 13C NMR
0
0
0
0
0
0
0
0
H4 /H5 ), 2.38 (s, 1H, CH3), 2.65–2.52 (m, 2H, H4 /H5 ), 2.95–2.82 (m,
(100.5 MHz, DMSO-d6): d = 21.9 (C4 /C5 ); 30.9 (C4 /C5 ), 48.9
0
0
0
0
1H, H4 /H5 ), 5.12 (dd, J = 12.6, 5.4 Hz, 1H, H3 ), 11.10 (s, 1H, NH);
(CH3 ), 122.7 (Ar-CH), 127.0 (Ar-C), 127.1 (2 ꢂ Ar-CH), 129.3
13C NMR (100.5 MHz, DMSO-d6): d = 48.9 (C3 ), 122.6, 128.0,
(2 ꢂ Ar-CH), 132.4 (Ar-C), 134.8 (Ar-CH), 134.2 (Ar-C), 136.3 (Ar-
0
128,6, 129.3, 129.7, 131.8, 132.5, 132.9, 134.2, 136.3, 138.1,
139.4, (C40, C50, CH3), 166.6 (C@O), 166.8 (C@O), 169.8
(C@O)172.8 (C@O); IR (neat): 3220 (N–HN–H), 1710 (C@O),
1612, 1348, 1262, 1198, 813, 731, 681 cmꢀ1; EI-MS (m/z) = 348
(100) [M]Å+, 238 (26), 165 (33); EI-HRMS calcd for C20H16N2O4:
348.1110; found: 348.1113.
CH), 138.5 (Ar-C), 139.3 (Ar-C), 166.4 (C@O), 166.7 (C@O), 167.4
~
(C@O), 169.8 (C@O), 172.7 (C@O); IR (KBr)
m
: 3422 (N–H), 1710
(C@O), 1390, 1208, 746 cmꢀ1; EI-HRMS calcd for C20H15N3O5:
377.1011; found: 377.1005.
7.3.13. (R,S)-2-(2,6-Dioxopiperidin-3-yl)-4-(4-isopropoxyphenyl)-
isoindoline-1,3-dione (32)
7.3.10. (R,S)-2-(2,6-Dioxopiperidin-3-yl)-4-(4-isobutylphenyl)iso-
indoline-1,3-dione (29)
Compound 32 was prepared using the Suzuki general protocol
Method C using 4-isopropoxyphenylboronic acid. Flash column
chromatography (7:20 ethyl acetate/hexane) afforded the title
biaryl compound 32 as a bright yellow solid (71%); mp = 100–
103°C; Rf = 0.62 (1:1 ethyl acetate/hexane); 1H NMR (400 MHz,
DMSO-d6): d = 1.29 (d, J = 6.0 Hz, 6H, 2 ꢂ CH3), 2.03–2.06 (m, 1H,
Compound 29 was prepared using Suzuki general protocol
Method C using 4-isobutylphenylboronic acid 21. Flash column
chromatography (7:20 ethyl acetate/hexane) gave the title biaryl
compound 29 as a light yellow solid (91%). mp = 120–122°C;
Rf = 0.27 (7:20 ethyl acetate/hexane); 1H NMR (400 MHz, DMSO-
d6): d = 0.87 (s, 6H, 2 ꢂ CH3), 1.86–1.90 (m, 1H, CH), 2.03–2.06
0
0
0
0
0
0
H4 /H5 ), 2.50–2.61 (m, 2H, H4 /H5 ), 2.83–2.88 (m, 1H, H4 /H5 ),
4.69 (septet, J = 6.0 Hz, 1H, CH(CH3)2), 5.11 (dd, J = 12.8 and
0
0
0
0
00
00
(m, 1H, H4 /H5 ), 2.49–2.50 (m, 2H, CH2), 2.52–2.60 (m, 2H, H4 /
5.2 Hz, 1H, H3 ), 7.00 (‘d’, ‘J’ = 8.4 Hz, 2H, H3 /H5 ), 7.52 (‘d’,
0
0
0
00
00
H5 ), 2.83–2.87 (m, 1H, H4 /H5 ), 5.11 (dd, J = 12.8 and 4.4 Hz, 1H,
‘J’ = 8.4 Hz, 2H, H2 /H6 ), 7.77 (m, 1H, Ar-CH), 7.88 (m, 2H, Ar-CH),
H3 ), 7.25 (‘d’, ‘J’ = 6.4 Hz, 2H, H3 /H5 ), 7.50 (‘d’, ‘J’ = 6.4 Hz, 2H,
11.09 (s, 1H, NH); 13C NMR (100.5 MHz, DMSO-d6): d = 21.8
0
00
00
00
00
0
0
0
0
0
H2 /H6 ), 7.80 (m, 1H, Ar-CH), 7.91 (m, 2H, Ar-CH), 11.09 (s, 1H,
(2 ꢂ CH3), 21.9 (C4 /C5 , CH2), 30.9 (C4 /C5 , CH2), 48.8 (C3 , CH),
69.2 (CH), 114.8 (2 ꢂ Ar-CH), 121.7 (Ar-CH), 126.3 (Ar-C), 127.6
(Ar-C), 130.9 (2 ꢂ Ar-CH), 132.5 (Ar-C), 134.6 (Ar-CH), 136.3 (Ar-
CH), 140.1 (Ar-C), 157.9 (Ar-CO), 166.7 (C@O), 166.8 (C@O), 169.8
~
(C@O), 172.7 (C@O); IR (KBr,) m: 3234 (N–H), 2976, 1770, 1712,
13
0
0
NH);
C NMR (100.5 MHz, DMSO-d6): d = 22.4 (C4 /C5 ), 22.6
0
0
0
(2 ꢂ CH3), 30.0 (CH), 31.6 (C4 /C5 ), 44.5 (CH2), 49.3 (C3 ), 122.6
(Ar-CH), 127.1 (Ar-C), 129.0 (2 ꢂ Ar-CH), 129.7 (2 ꢂ Ar-CH), 132.9
(Ar-C), 133.6 (Ar-C), 135.1 (Ar-CH), 136.8 (Ar-CH), 140.7 (Ar-C),
142.2 (Ar-C), 167.1 (C@O), 167.2 (C@O), 170.3 (C@O), 173.2
~
1393, 1189 (C–O), 749 cmꢀ1; MS EI, m/z (%): 392 (37) [M]Å+, 350
(100) [MꢀC3H7]Å+, 278 (15), 265 (24); EI-HRMS calcd for
C22H20N2O5: 392.1372; found: 392.1364.
(C@O); IR (KBr, cmꢀ1
) m: 3418 (N–H), 2955, 1714 (C@O), 1393,
1198; MS EI, m/z (%): 390 (61) [M]Å+, 348 (53), 347 (100)
[MꢀC3H7]Å+, 275 (16), 262 (15); EI-HRMS calcd for C23H22N2O4:
390.1580; found: 390.1584.
7.3.14. (R,S)-4-(4-Acetylphenyl)-2-(2,6-dioxopiperidin-3-yl)iso-
indoline-1,3-dione (33)
7.3.11. (R,S)-2-(2,6-Dioxopiperidin-3-yl)-4-(4-hydroxyphenyl)iso-
indoline-1,3-dione (30)
Compound 33 was prepared using the Suzuki general protocol
Method B using 4-acetylphenylboronic acid 20. Flash column chro-
matography (2:25 methanol/dichloromethane) and additional
recrystallisation from ethyl acetate to afforded the title biaryl com-
pound 33, as a white solid (55%); mp = >230°C; Rf = 0.3 (2:25 meth-
anol/dichloromethane); 1H NMR (300 MHz, DMSO-d6): d = 2.04–
Compound 30 was prepared using the Suzuki general protocol
Method C using 4-hydroxyphenylboronic acid. In this instance
the reaction mixture was then washed with HCl (1 M, 10 mL)
and the organic layer dried (MgSO4) and fused to silica before being
purified via flash column chromatography (11:20 ethyl acetate/
hexane) to afford the desired biaryl compound 30, as a yellow solid
(51%); mp = 215–217 °C; Rf = 0.25 (1:1 ethyl acetate/hexane); 1H
0
0
0
0
2.08 (m, 1H, H4 /H5 ), 2.48–2.56 (m, 2H, H4 /H5 ), 2.64 (s, 3H, CH3),
0
0
0
2.81–2.88 (m, 1H, H4 /H5 ), 5.12 (dd, J = 12.9 and 5.4 Hz, 1H, H3 ),
00
00
7.73 (‘d’, ‘J’ = 8.4 Hz, 2H, H2 /H6 ), 7.85 (dd, J = 6.3 and 2.7 Hz, 1H,
0
0
00
NMR (400 MHz, DMSO-d6): d = 2.03–2.05 (m, 1H, H4 /H5 ), 2.53–
Ar-CH), 7.95–7.99 (m, 2H, Ar-CH), 8.04 (‘d’, ‘J’ = 8.7 Hz, 2H, H3
00
/
2.61 (m, 2H, H4 /H5 ), 2.84–2.92 (m, 1H, H4 /H5 ), 5.11 (dd, J = 12.8
H5 ), 11.10 (s, 1H, NH); 13C NMR (75.5 MHz, DMSO-d6): d 21.9
0
0
0
0
0
00
00
0
0
0
0
0
and 5.2 Hz, 1H, H3 ), 6.84 (‘d’, ‘J’ = 8.0 Hz, 2H, H3 /H5 ), 7.43 (‘d’,
(C4 /C5 ), 26.8 (CH3) 30.9 (C4 /C5 ), 48.9 (C3 ), 123.0 (Ar-CH), 127.1
(Ar-C), 127.8 (2 ꢂ Ar-CH), 129.8 (2 ꢂ Ar-CH), 132.4 (Ar-C), 134.8
(Ar-CH), 136.2 (Ar-C), 136.5 (Ar-C), 138.9 (Ar-C), 140.4 (Ar-C),
166.4 (C@O), 166.6 (C@O), 169.8 (C@O), 172.8 (C@O), 197.7
~
(C@O); IR (KBr) m: 3462 (N–H), 1773, 1714 (C@O), 1393, 1268,
00
00
‘J’ = 8.0 Hz, 2H, H2 /H6 ), 7.75 (‘d’, ‘J’ = 6.8 Hz, 1H, Ar-CH), 7.85 (‘d’,
‘J’ = 5.1 Hz, 2H, Ar-CH), 9.74 (s, 1H, OH), 11.09 ppm (s, 1H, NH);
C NMR (100.5 MHz, DMSO-d6): d = 21.9 (C4 /C5 , CH2), 30.9 (C4 /
13
0
0
0
0
0
C5 , CH2), 48.8 (C3 , CH), 114.8 (2 ꢂ Ar-CH), 121.5, 126.1, 126.3,
130.9, 132.5, 134.6, 136.2, 140.6, 158.1 (Ar-CO), 166.7 (C@O),
1200 cmꢀ1; MS EI, m/z (%): 376 (38) [M]Å+, 261 (100) [MꢀCH3]Å+;
~
m
166.8 (C@O), 169.8 (C@O), 172.7 (C@O); IR (KBr)
: 3423 (N–H),
EI-HRMS calcd for C21H16N2O5: 376.1059; found: 376.1066.
1763, 1711 (C@O), 1396, 1199 (C–O), 750 cmꢀ1; MS EI, m/z (%):
350 (100) [M]Å+, 265 (24); EI-HRMS calcd for C19H14N2O5:
350.0902; found: 350.0911.
7.3.15. (R,S)-2-(2,6-Dioxopiperidin-3-yl)-5-phenylisoindole-1,3-
dione (34)
Compound 34 was prepared using the Suzuki general protocol
Method C using phenylboronic acid. Column chromatography
(2:3 ethyl acetate/hexane) afforded the desired 34 as a pale yellow
solid (91%); mp; >230 °C; Rf = 0.3 (1:1 ethyl acetate/hexane); 1H
7.3.12. (R,S)-4-(2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-
4-yl)benzamide (31)
Compound 31 was prepared using the Suzuki general protocol
Method C using 4-aminocarbonylphenylboronic acid. Flash column
chromatography (1:9 methanol/dichloromethane) and recrystalli-
sation from acetonitrile to afford the desired biaryl compound 31
as a white solid (12% yield); mp = >230°C; Rf = 0.37 (1:1 acetone/
toluene); 1H NMR (400 MHz, DMSO-d6): d = 2.06–2.08 (m, 1H,
0
0
NMR (400 MHz, DMSO-d6): d = 2.13–2.05 (m, 1H, H4 /H5 ), 2.66–
0
0
0
0
2.53 (m, 2H, H4 /H5 ), 2.97–2.85 (m, 1H, H4 /H5 ), 5.19 (dd, J = 13.0,
5.4 Hz, 1H, H3 ), 8.31–7.30 (Ar-H), 11.14 (s, 1H, NH); 13C NMR
0
0
0
0
0
(100.5 MHz, DMSO-d6): d = 22.0 (C4 /C5 , CH2), 31.0 (C4 /C5 , CH2),
0
49.1 (CH3 , CH), 121.4, 124.1, 127.3, 127.4, 129.4, 129.9, 130.0,
0
0
0
0
0
0
H4 /H5 ), 2.48–2.61 (m, 2H, H4 /H5 ), 2.84–2.88 (m, 1H, H4 /H5 ),
132.3, 133.0, 134.0, 138.1, 146.7, 167.0 (C@O), 167.0 (C@O),
169.9 (C@O), 172.8 (C@O); IR (neat): 3447 (N–H), 1713 (C@O),
1380, 1261, 1198, 1116, 743, 700 cmꢀ1; MS (m/z) = 334 (100)
0
5.12 (dd, J = 12.8 and 5.2 Hz, 1H, H3 ), 7.45 (s, 1H, NH2), 7.66 (‘d’,
‘J’ = 8.0 Hz, 2H, H2 /H6 ), 7.83–7.85 (m, 1H, Ar-CH), 7.92–7.96 (m,
00
00

660
S. G. Stewart et al. / Bioorg. Med. Chem. 18 (2010) 650–662
[M]Å+, 249 (59), 224 (31), 180 (26), 152 (31), 102 (32); HRMS calcd
for C19H14N2O4: 334.0954; found: 334.0959.
7.3.19. (R,S)-2-(2,6-Dioxopiperidin-3-yl)-5-(4-isopropoxyphenyl)-
isoindoline-1,3-dione (38)
Compound 38 was prepared using the Suzuki general protocol
Method C using 4-isopropoxyphenylboronic acid. Flash column
chromatography (7:20 ethyl acetate/hexane) afforded the title
biaryl compound 38 as a bright yellow solid (78%); mp = 105–
108°C; Rf = 0.26 (7:20 ethyl acetate/hexane); 1H NMR (400 MHz,
DMSO-d6): d = 1.30 (d, J = 6.0 Hz, 6H, 2 ꢂ CH3), 2.07–2.09 (m, 1H,
7.3.16. (R,S)-2-(2,6-Dioxopiperidin-3-yl)-5-p-tolylisoindole-1,3-
dione (35)
Compound 35 was prepared using the Suzuki general protocol
Method C using 4-toluyl pinacolboronic acid ester. Column chro-
matography (2:3 ethyl acetate/hexane) and subsequently recrys-
tallised from ethyl acetate to afford the desired biaryl compound
0
0
0
0
0
0
H4 /H5 ), 2.50–2.64 (m, 2H, H4 /H5 ), 2.82–2.99 (m, 1H, H4 /H5 ),
4.71 (septet, J = 6.0 Hz, 1H, CH(CH3)2), 5.17 (dd, J = 12.8 and
35 as an off-white solid (38%); Rf = 0.2 (2:5 ethyl acetate/hexane);
1
0
00
00
5.2 Hz, 1H, H3 ), 7.05 (‘d’, ‘J’ = 8.8 Hz, 2H, H3 /H5 ), 7.78 (‘d’,
0
0
H NMR (400 MHz, DMSO-d6): d = 2.13–2.03 (m, 1H, H4 /H5 ), 2.37
00
00
‘J’ = 9.2 Hz, 2H, H2 /H6 ), 7.95 (‘d’, ‘J’ = 5.2 Hz, 1H, Ar-CH), 8.12
0
0
0
(s, 3H, CH3), 2.66–2.52 (m, 2H, H4 /H5 ), 2.97–2.83 (m, 1H, H4 /
(‘d’, ‘J’ = 5.2 Hz, 2H, Ar-CH), 11.14 (s, 1H, NH); 13C NMR
0
0
H5 ), 5.18 (dd, J = 13.0 and 5.8 Hz, 1H, H3 ), 8.15–7.12 (Ar-H),
11.14 (s, 1H, NH); 13C NMR (100.5 MHz, DMSO-d6): d = 31.0–20.7
0
0
(100.5 MHz, DMSO-d6): d = 21.8 (2 ꢂ CH3), 22.0 (C4 /C5 , CH2),
0
0
0
0
0
30.9 (C4 /C5 , CH2), 49.0 (C3 , CH), 69.4 (CH), 116.1 (2 ꢂ Ar-CH),
120.6 (Ar-CH), 124.0 (Ar-CH), 128.7 (2 ꢂ Ar-CH), 128.9 (Ar-C),
129.9 (Ar-C), 132.0 (Ar-CH), 132.7 (Ar-C), 146.4 (Ar-C), 158.4
(Ar-CO), 167.0 (C@O), 167.1 (C@O), 169.9 (C@O), 172.8 (C@O);
~
0
(C4 , C5 , CH3), 49.1 (C3 , CH), 121.1, 124.1, 127.1, 128.0, 129.5,
129.8, 132.3, 132.6, 134.2, 138.7, 139.3, 146.6, 167.0 (C@O),
167.0 (C@O), 169.9 (C@O), 172.8 (C@O); IR (NaCl): 1707 (C@O),
1612, 1349, 1260, 1182, 816, 731, 681 cmꢀ1; MS (m/z) = 348
(100) [M]Å+, 263 (47), 238 (21), 166 (21); EI-HRMS calcd for
C20H16N2O4: 348.1110; found: 348.1118.
IR (KBr)
m: 3247 (N–H), 2976, 1774, 1716 (C@O), 1384, 1187
(C–O) cmꢀ1; EI MS, m/z (%): 392 (41) [M]Å+, 349 (100), [MꢀC2H7]Å+,
264 (33), 238 (58); EI-HRMS calcd for C22H20N2O5: 392.1372;
found: 392.1378.
7.3.17. (R,S)-2-(2,6-Dioxopiperidin-3-yl)-5-(4-hydroxyphenyl)-
isoindoline-1,3-dione (36)
7.3.20. (R,S)-4-(3,3-Dimethylbut-1-ynyl)-2-(2,6-dioxopiperidin-
3-yl)isoindoline-1,3-dione (39)
Compound 36 was prepared using the Suzuki general protocol
Method C using 4-hydroxyphenylboronic acid. The reaction mix-
ture was then transferred to a separatory funnel and washed with
an aqueous HCl solution (1 M, 10 mL), brine (10 mL) and dried
(MgSO4). The organic mixture was then fused to silica before being
purified via flash column chromatography (11:20 ethyl acetate/
hexane) to afford the title biaryl compound 36, as a bright yellow
Compound 39 was prepared using general protocol for the
Sonogashira reaction using 3,3-dimethyl-1-butyne. Flash column
chromatography (3:7 ethyl acetate/hexane) afforded the alkyne
compound 39, as a yellow solid (60%); mp = 228–230°C; Rf = 0.29
(1:1 ethyl acetate/hexane); 1H NMR (400 MHz, DMSO-d6):
0
0
d = 1.34 (s, 9H, 3 ꢂ CH3), 2.04–2.08 (m, 1H, H4 /H5 ), 2.52–2.62 (m,
solid (91%); mp = >230°C; Rf = 0.31 (11:20 ethyl acetate/hexane);
0
0
0
0
2H, H4 H5 ), 2.85–2.88 (m, 1H, H4 H5 ), 5.15 (dd, J = 12.8 and
1
0
0
H NMR (400 MHz, DMSO-d6): d = 2.05–2.09 (m, 1H, H4 /H5 ),
5.2 Hz, 1H, H3 ), 7.76–7.87 (m, 3H, Ar-CH), 11.13 (s, 1H, NH); 13C
0
0
0
0
0
2.55–2.63 (m, 2H, H4 /H5 ), 2.81–2.99 (m, 1H, H4 /H5 ), 5.17 (dd,
0
0
NMR (100.5 MHz, DMSO-d6): d = 22.4 (C4 /C5 , CH2), 28.4
0
00
00
J = 12.6 and 5.1 Hz, 1H, H3 ), 6.90 (‘d’, ‘J’ = 8.4 Hz, 2H, H3 /H5 ),
0
0
0
(C(CH3)3), 30.7 (3 ꢂ CH3), 31.4 (C4 /C5 , CH2), 49.4 (C3 , CH), 75.1
(C„C), 106.6 (C„C), 120.4 (Ar-C), 123.0 (Ar-H), 130.7 (Ar-C),
132.5 (Ar-C), 135.0 (Ar-H), 138.1 (Ar-H), 166.1 (C@O), 166.7
~
(C@O), 170.3 (C@O), 173.2 (C@O); IR (KBr) m: 3440 (N–H), 2222
00
00
7.71 (‘d’, ‘J’ = 8.4 Hz, 1H, H2 /H6 ), 7.92 (‘d’, ‘J’ = 6.3 Hz, 2H, Ar-CH),
8.08 (‘d’, ‘J’ = 5.1 Hz, 2H, Ar-CH), 9.86 (s, 1H, OH), 11.15 (s, 1H,
13
0
0
NH); C NMR (75.5 MHz, DMSO-d6): d = 22.0 (C4 /C5 , CH2), 31.0
0
0
0
(C4 /C5 , CH2), 49.0 (C3 ), 116.1 (2 ꢂ Ar-CH), 120.4 (Ar-CH), 124.0
(Ar-CH), 128.7 (2 ꢂ Ar-CH), 131.8 (2 ꢂ Ar-CH), 132.4 (2 ꢂ Ar-C),
146.7 (Ar-C), 158.6 (Ar-CO), 167.0 (C@O), 167.1 (C@O), 169.9
~
(C„C), 1717 (C@O), 1391, 1202, 746 cmꢀ1; MS EI, m/z (%): 339
(42) [M]Å+, 323 (98), 250 (45), 269 (70); EI-HRMS calcd for
C19H18N2O4: 338.1266; found: 338.1267.
(C@O), 172.8 (C@O); IR (KBr, cmꢀ1
) m: 3222 (N–H), 1773, 1725
(C@O), 1607, 1333, 1200 (C–O), 828; MS EI, m/z (%): 351 (22)
[M+H]Å+, 350 (100) [M]Å+, 265 (35), 239 (56) [MꢀC5H6NO2]Å+; EI-
HRMS calcd for C19H14N2O5: 350.0902; found: 350.0909.
7.3.21. (R,S)-2-(2,6-Dioxopiperidin-3-yl)-5-phenylethynyliso-
indole-1,3-dione (40)
Compound 40 was prepared using general protocol for the
Sonogashira reaction using phenylacetylene. Flash column chro-
matography (1:1 ethyl acetate/hexane) and subsequently recrys-
tallisation from ethyl acetate afforded 40 as a yellow solid
(59%); Rf = 0.3 (1:1 ethyl acetate/hexane); 1H NMR (400 MHz,
7.3.18. (R,S)-4-(2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-
-5-yl)benzamide (37)
Compound 37 was prepared using the Suzuki general protocol
Method C using 4-aminocarbonylphenylboronic acid. The crude
mixture was run through a short silica plug eluting with ethyl ace-
tate, before being recrystallised from acetonitrile to afford the title
biaryl amide 37 as a tan solid (37%); mp = >230°C; Rf = 0.35 (7:20
acetone/toluene); 1H NMR (300 MHz, DMSO-d6): d = 2.07–2.11
0
0
0
0
CDCl3): d = 2.23–2.13 (m, 1H, H4 /H5 ), 2.96–2.70 (m, 3H, H4 /H5 ),
0
5.00 (dd, J = 12.6, 5.4 Hz, 1H, H3 ), 7.42–7.37 (m, 3H, Ar-H),
7.59–7.54 (m, 2H, Ar-H), 7.88–7.85 (m, 2H, Ar-H), 8.29 (s, 1H,
NH), 8.01–7.95 (m, 1H, Ar-H); 13C NMR (100.5 MHz, CDCl3):
0
0
0
0
0
d = 22.7 (C4 /C5 , CH2), 31.6 (C4 /C5 , CH2), 49.6 (C3 ), 87.7 (C„C),
94.6 (C„C), 122.1 (Ar-C), 123.9 (Ar-CH)126.7 (Ar-CH), 128.7
(Ar-CH), 129.5 (Ar-CH), 130.4 (Ar-C), 132.0 (Ar-CH), 132.1 (Ar-
C), 137.4 (Ar-CH), 166.8 (C@O), 166.8 (C@O), 168.0 (C@O),171.0
(C@O); IR (neat): 3446 (N–H), 2213 (C„C), 1717 (C@O), 1380,
1196, 746 cmꢀ1; MS (m/z) = 358 (100) [M]Å+, 273 (40), 176 (24),
126 (26); EI-HRMS calcd for C21H14N2O4: 358.0954; found:
358.0952.
0
0
0
0
0
(m, 1H, H4 /H5 ), 2.50–2.65 (m, 2H, H4 /H5 ), 2.82–3.00 (m, 1H, H4 /
0
0
H5 ), 5.20 (dd, J = 12.6 and 5.4 Hz, 1H, H3 ), 7.46 (s, 1H, NH2), 7.95
00
00
(‘d’, ‘J’ = 8.7 Hz, 2H, H2 /H6 ), 8.01–8.04 (m, 3H, Ar-CH), 8.10 (s,
1H, NH2), 8.24 (‘d’, ‘J’ = 6.3 Hz, 2H, H3 /H5 ), 11.15 (s, 1H, NH); 13C
00
00
0
0
0
0
NMR (75.5 MHz, DMSO-d6): d = 22.0 (C4 /C5 ), 30.9 (C4 /C5 ), 49.1
0
(CH3 ), 121.7 (Ar-CH), 124.1 (Ar-CH), 127.3 (2 ꢂ Ar-CH), 128.3
(2 ꢂ Ar-CH), 130.3 (Ar-C), 132.3 (Ar-C), 133.2 (Ar-CH), 134.4 (Ar-
C), 140.3 (Ar-C), 145.7 (Ar-C), 166.8 (C@O), 166.9 (C@O), 167.2
~
(C@O), 169.8 (C@O), 172.8 (C@O); IR (KBr)
m
: 3455 (N–H), 1709
7.3.22. (R,S)-2-(2,6-Dioxopiperidin-3-yl)-4-(3-hydroxy-3-methyl-
but-1-ynyl)isoindoline-1,3-dione (41)
Compound 41 was prepared using general protocol for the
Sonogashira reaction using 2-methyl-3-butyn-2-ol. Flash column
(C@O), 1390, 1203, 746 cmꢀ1; MS EI, m/z (%): 377 (100) [M]Å+,
332 (10) [MꢀCONH2]Å+, 292 (43), 265 (33); EI-HRMS calcd for
C20H15N2O5: 377.1012; found: 377.1010.

S. G. Stewart et al. / Bioorg. Med. Chem. 18 (2010) 650–662
661
13
chromatography (2:3 ethyl acetate/hexane) afforded the title al-
kyne 41, as straw yellow solid (64%); mp = 116–118°C;
Rf = 0.28 (2:3 ethyl acetate/hexane); 1H NMR (400 MHz, DMSO-
1H, NH); C NMR (100.5 MHz, CDCl3): 22.8 (C40/C50), 31.6 (C40/
C50), 49.5 (C30), 84.5 (C„C), 97.6 (C„C), 121.2 (Ar-C), 122.3 (Ar-
C), 123.2 (Ar-CH), 128.6 (Ar-CH), 129.5 (Ar-CH), 132.3 (Ar-CH),
134.1 (Ar-CH), 138.1 (Ar-CH), 166.0 (C@O), 166.6 (C@O), 167.9
(C@O), 170.8 (C@O); IR (NaCl): 3275 (N–H), 2214 (C„C), 1711
(C@O), 1388, 1203, 745 cmꢀ1; MS (m/z) = 358 (100) [M]Å+, 273
(20), 176 (21); EI-HRMS calcd for C21H14N2O4: 358.0954; found:
358.0954.
a
0
0
d6): d = 1.51 (s, 6H, 2 ꢂ CH3), 2.03–2.10 (m, 1H, H4 /H5 ), 2.50–
0
0
0
0
2.64 (m, 2H, H4 /H5 ), 2.83–2.93 (m, 1H, H4 /H5 ), 5.15 (dd,
0
J = 12.2 and 5.6 Hz, 1H, H3 ), 5.58 (s, 1H, OH), 7.79–7.90 (m,
3H, Ar-H), 11.15 (s, 1H, N–H); 13C NMR (100.5 MHz, DMSO-d6):
0
0
0
0
d = 21.9 (C4 /C5 , CH2), 30.9 (C4 /C5 CH2), 21.3 (2 ꢂ CH3), 48.9
0
(C3 , CH), 63.8 (COH), 75.9 (C„C), 103.2 (C„C), 119.3 (Ar-C),
122.9 (Ar-CH), 130.3 (Ar-C), 132.0 (Ar-C), 134.7 (Ar-CH), 137.9
7.3.26. (R,S)-2-(2,6-Dioxopiperidin-3-yl)-5-(3-hydroxy-3-
methylbut-1-ynyl)isoindoline-1,3-dione (45)
(Ar-CH), 165.6 (C@O), 166.3 (C@O), 169.8 (C@O), 172.7 (C@O);
ꢀ1
~
m
IR (KBr)
: 3421 (O–H), 1718 (C@O), 1394, 1198 (O–H) cm
;
Compound 45 was prepared using general protocol for the
Sonogashira reaction using 2-methyl-3-butyn-2-ol. Flash column
chromatography (4:1 ethyl acetate/hexane) afforded the alkyne
45, as a yellow solid (76%). Mp = 62–65°C; Rf = 0.28 (4:1 ethyl ace-
tate/hexane); 1H NMR (400 MHz, DMSO-d6): d = 1.51 (s, 6H,
MS EI, m/z (%): 340 (100) [M]Å+, 323 (100) [MꢀCH3]Å+, 262 (28),
237 (21); EI-HRMS calcd for C18H16N2O5: 340.1059; found:
340.1061.
7.3.23. (R,S)-6-(2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-
4-yl)hex-5-ynenitrile (42)
0
0
0
0
2 ꢂ CH3), 2.04–2.08 (m, 1H, H4 /H5 ), 2.50–2.62 (m, 2H, H4 /H5 ),
0
0
0
2.85–2.88 (m, 1H, H4 /H5 ), 5.15 (dd, J = 12.8 and 5.2 Hz, 1H, H3 ),
5.58 (s, 1H, OH), 7.79–7.90 (m, 3H, Ar-H), 11.12 (s, 1H, NH); 13C
Compound 42 was prepared using general protocol for the
Sonogashira reaction using 5-hexynylnitrile. Flash column chroma-
tography (1:1 ethyl acetate/hexane) and recystallisation from ace-
tonitrile afforded the title alkynyl compound 42 as a white solid
(21%). mp = 191–193°C. Rf = 0.33 (1:1 ethyl acetate/hexane); 1H
0
0
0
0
NMR (100.5 MHz, DMSO-d6): d = 21.5 (C4 /C5 , CH2), 30.5 (C4 /C5 ,
0
CH2), 30.8 (2 ꢂ CH3), 48.5 (C3 , CH), 63.4 (COH), 75.5 (C„C), 102.8
(C„C), 118.9 (Ar-C), 122.5 (Ar-CH), 129.8 (Ar-C), 131.6 (Ar-C),
134.3 (Ar-CH), 137.5 (Ar-CH), 165.2 (C@O), 165.8 (C@O), 169.4
00
NMR (300 MHz, DMSO-d6): d = 1.90 (t, J = 7.2 Hz, 2H, H4 ), 2.03–
~
(C@O), 172.3 (C@O); IR (KBr,)
m
: 3415 (O–H), 2980 (N–H), 2226
(C„C), 1776, 1716 (C@O), 1384, 1201 (C–O) cmꢀ1; MS EI, m/z
(%): 340 (10) [M]Å+, 325 (100) [MꢀCH3]Å+, 262 (90); EI-HRMS calcd
for C18H16N2O5: 340.1059; found: 340.1044.
0
0
0
0
2.09 (m, 1H, H4 /H5 ), 2.51–2.60 (m, 2H, H4 /H5 ), 2.66 (t,
00
00
0
J = 6.7 Hz, 2H, H5 ), 2.77 (t, J = 7.2 Hz, 2H, H3 ), 2.90 (m, 1H, H4 /
0
0
H5 ), 5.16 (dd, J = 12.6 and 5.1 Hz, 1H, H3 ), 7.83–7.90 (m, 3H, Ar-
CH), 11.13 (s, 1H, NH); 13C NMR (75.5 MHz, DMSO-d6): d = 15.3
00
00
0
0
00
(C4 , CH2), 18.2 (C3 , CH2), 21.9 (C4 /C5 , CH2), 23.8 (C5 , CH2), 30.9
7.3.27. (R,S)-2-(2,6-Dioxopiperidin-3-yl)-5-(3-(methylamino)-
prop-1-ynyl)isoindoline-1,3-dione (46)
0
0
0
(C4 /C5 , CH2), 49.0 (C3 , CH), 77.2 (C„C), 96.5 (C„C), 119.5
(C„N), 120.2 (Ar-C), 122.9 (Ar-CH), 130.3 (Ar-C), 132.0 (Ar-C),
134.7 (Ar-C), 138.2 (Ar-CH), 165.9 (C@O), 166.3 (C@O), 169.8
~
Compound 46 was prepared using general protocol for the
Sonogashira reaction using N-methylpropargylamine. Flash col-
umn chromatography (1:9 methanol/dichloromethane) afforded
the alkyne 46, as a brown solid (21%); mp = >230°C; Rf = 0.31
(1:9 methanol/dichloromethane); 1H NMR (400 MHz, DMSO-d6):
(C@O), 172.8 (C@O); IR (KBr)
m: 3425 (N–H), 2922, 2246, 1715
(C@O), 1391, 1203, 744 cmꢀ1; MS EI, m/z (%): 349 (12) [M]Å+, 321
(100) [MꢀH2CN]Å+, 235 (25); EI-HRMS calcd for C19H15N3O4:
349.1063; found: 349.1071.
0
0
d = 2.01–2.08 (m, 1H, H4 /H5 ), 2.39 (s, 3H, CH3), 2.50–2.59 (m, 2H,
0
0
0
0
H4 /H5 ), 2.84–2.95 (m, 1H, H4 /H5 ), 3.58 (s, 2H, CH2), 5.16 (dd,
7.3.24. (R,S)-2-(2,6-Dioxopiperidin-3-yl)-4-(3-hydroxyprop-1-
ynyl)isoindoline-1,3-dione (43)
0
J = 12.8 and 5.6 Hz, 1H, H3 ), 7.84–7.92 (m, 3H, Ar-H), 11.12 (brs,
1H, NH); C NMR (100.5 MHz, DMSO-d6): d = 21.9 (C4 /C5 , CH2),
13
0
0
Compound 43 was prepared using general protocol for the
Sonogashira reaction using propargyl alcohol. Column chromatog-
raphy (1:3 ethyl acetate/hexane) afforded the title alkyne 43, as a
dark yellow solid (78%); mp = 214–217°C; Rf = 0.21 (1:3 ethyl ace-
tate/hexane); 1H NMR (400 MHz, DMSO-d6): d = 2.00–2.05 (m, 1H,
0
0
0
30.9 (C4 /C5 , CH2), 34.7 (CH3), 49.1 (C3 , CH), 81.8 (C„C), 93.6
(C„C), 123.8 (Ar-CH), 125.6 (Ar-CH), 129.0 (Ar-C), 130.1 (Ar-C),
132.8 (Ar-C), 137.4 (Ar-CH), 166.4 (C@O), 166.5 (C@O), 169.8
~
(C@O), 172.7 (C@O); IR (KBr) m: 3423 (N–H), 2224 (C„C), 1776,
1718 (C@O), 1383, 1204, 1117 cmꢀ1; MS EI, m/z (%): 324 (100)
[M]Å+, 262 (79), 239 (19); EI-HRMS calcd for C17H15N3O4:
324.0984; found: 324.0989.
0
0
0
0
0
0
H4 /H5 ), 2.50–2.81 (m, 2H, H4 /H5 ), 2.81–2.88 (m, 1H, H4 /H 5), 4.35
0
(d, J = 5.6 Hz, 2H, CH2OH), 5.10 (dd, J = 12.4 and 5.2 Hz, 1H, H3 ),
5.44 (t, J = 5.6 Hz, 1H, OH), 7.83–7.87 (m, 4H, Ar-CH), 11.09 (s,
13
0
0
1H, NH); C NMR (100.5 MHz, DMSO-d6): d = 22.3 (C4 /C5 , CH2),
0
0
0
7.3.28. (R,S)-6-(2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-
5-yl)hex-5-ynenitrile (47)
31.3 (C4 /C5 , CH2), 49.4 (C3 , CH), 49.9 (CH2OH), 79.3 (C„CCH2OH),
97.6 (C„CCH2OH), 119.5 (Ar-C), 123.6 (Ar-CH), 130.6 (Ar-C), 132.5
(Ar-C), 135.2 (Ar-CH), 138.7 (Ar-CH), 166.0 (C@O), 166.6 (C@O),
~
Compound 47 was prepared using general protocol for the
Sonogashira reaction using 5-hexynylnitrile. Flash column chroma-
tography (1:5?9:20 ethyl acetate/hexane) afforded the title alky-
nyl compound 47, as a light yellow solid (64%); mp = 180–182°C.
Rf = 0.26 (9:20 ethyl acetate/hexane); 1H NMR (300 MHz, DMSO-
170.2 (C@O), 173.1 (C@O); IR (KBr) m: 3464 (O–H), 3283 (N–H),
2225 (C„C), 1774, 1711 (C@O), 1384, 1206, 1037, 748 cmꢀ1; MS
EI, m/z (%): 312 (23) [M]Å+, 284 (28), 283 (100), 200 (24), 198
(33), 184 (20); EI-HRMS calcd for C16H11N2O5: 312.0746; found:
312.0751.
00
0
0
d6): d = 1.89 (t, J = 6.9 Hz, 2H, H4 ), 2.04 (m, 1H, H4 /H5 ), 2.52–
0
0
00
00
0
0
2.72 (m, 6H, H4 /H5 , H3 , H5 ), 2.94 (m, 1H, H4 /H5 ), 5.15 (dd,
0
J = 12.6 and 5.1 Hz, 1H, H3 ), 7.91–7.95 (m, 3H, Ar-CH), 11.14 (s,
7.3.25. (R,S)-2-(2,6-Dioxopiperidin-3-yl)-4-phenylethynyliso-
indole-1,3-dione (44)
1H, NH); 13C NMR (75.5 MHz, DMSO-d6): d = 15.6 (C4 ), 18.1 (C3 ),
00
00
0
0
00
0
0
0
21.9 (C4 /C5 ), 23.7 (C5 ), 30.9 (C4 /C5 ), 49.1 (C3 ), 80.3 (C„C), 93.8
(C„C), 120.3 (C„N), 123.7 (Ar-CH), 125.8 (Ar-CH), 129.3 (Ar-C),
130.0 (Ar-C), 131.7 (Ar-C), 137.4 (Ar-CH), 166.4 (C@O), 166.5
~
(C@O), 169.7 (C@O), 172.7 (C@O); IR (KBr) m: 3417 (N–H), 2922,
Compound 44 was prepared using general protocol for the
Sonogashira reaction using phenylacetylene. Column chromatogra-
phy (1:10?1:1 ethyl acetate/hexane) afforded compound 44 (61%)
as
(400 MHz, CDCl3): d = 2.21–2.13 (m, 1H, H4 /H5 ), 2.97–2.69 (m,
a
tan solid; Rf = 0.2 (1:1 ethyl acetate/hexane); 1H NMR
2246 (C„C), 1389, 1204 cmꢀ1; MS EI, m/z (%): 349 (82) [M]Å+,
321 (15) [MꢀH2CN]Å+, 264 (100); EI-HRMS calcd for C19H15N3O4:
349.1063; found: 349.1068.
0
0
0
0
0
3H, H4 /H5 ), 5.02 (dd, J = 12.6, 5.4 Hz, 1H, H3 ), 7.41–7.37 (m, 2H,
Ar-H), 7.75–7.63 (m, 4H, Ar-H), 7.85–7.82 (m, 2H, Ar-H), 8.01 (s,

662
S. G. Stewart et al. / Bioorg. Med. Chem. 18 (2010) 650–662
2. (a) Ribatti, D.; Vacca, A. Leukemia 2005, 19, 1525–1531; (b) Melchert, M.; List,
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8.1. TNF reporter gene assay
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The effect of each compound on the inhibition of TNF expres-
sion and cellular viability was assessed using the FRT-Jurkat TNF
reporter cell line. Analysis was done using a FACSCalibur 4-colour
Flow Cytometer (Becton, Dickinson and Company, New Jersey,
USA). Data analysis was performed using FlowJo software (Tree-
Star, Ashland, OR, USA). Viable and non-viable cells present follow-
ing either solvent (DMSO) alone or compound treatment for 24 h,
were assayed using flow cytometry. Cells were counted by gating
on each cell population using a forward scatter (FSC) versus side
scatter (SSC) plot. Inhibition was determined as a decrease in
GFP fluorescence which was detected at a wavelength of 515 nm
on the FL3 channel of the instrument. The percentage of viable cells
was determined as the percentage of cells inside the FSC/SSC gate
that encompassed the major population in solvent-only treated
cells. Viability was confirmed following propidium iodide staining
and detection of flourescence at 570 nm on the FL2 channel. For
detection of cells undergoing apoptosis, following treatment with
each compound, cells were treated with fluorescein isothiocyanate
(FITC)-labeled substrate against cleaved caspase 3 using the Nuc-
view-488 caspase 3 assay (Biotium Inc., Hayward, USA). FITC fluo-
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PBMCs were isolated from leukocyte buffy coat (Australian Red
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were stimulated to express TNF using PMA (20 ng/mL) and ionomy-
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housekeeping gene b-actin as previously described.39 The thermal
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Statistical significance was determined by student’s unpaired t-test.
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The authors would like to thank the Cancer Council of Western
Australia for their generous support.
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Supplementary data
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References and notes
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as a Vital Medicine; Perseus, 2001.