Isosteric analogs of lenalidomide and pomalidomide: Synthesis and biological activity

Article abstract of DOI:10.1016/j.bmcl.2012.10.071

A series of analogs of the immunomodulary drugs lenalidomide (1) and pomalidomide (2), in which the amino group is replaced with various isosteres, was prepared and assayed for immunomodulatory activity and activity against cancer cell lines. The 4-methyl and 4-chloro analogs 4 and 15, respectively, displayed potent inhibition of tumor necrosis factor-α (TNF-α) in LPS-stimulated hPBMC, potent stimulation of IL-2 in a human T cell co-stimulation assay, and anti-proliferative activity against the Namalwa lymphoma cell line. Both of these analogs displayed oral bioavailability in rat.

Full text of DOI:10.1016/j.bmcl.2012.10.071

Bioorganic & Medicinal Chemistry Letters 23 (2013) 360–365
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Isosteric analogs of lenalidomide and pomalidomide: Synthesis
and biological activity
Alexander L. Ruchelman a, , Hon-Wah Man a, Weihong Zhang a, Roger Chen a, Lori Capone a, Jian Kang a,
⇑
Anastasia Parton a, Laura Corral a, Peter H. Schafer a, Darius Babusis b, Mehran F. Moghaddam b,
Yang Tang b, Michael A. Shirley b, George W. Muller a
a Drug Discovery Department, Celgene Corporation, 86 Morris Avenue, Summit, NJ 07901, United States
b Department of DMPK, Celgene Signal Research, 4550 Towne Centre Court, San Diego, CA 92121, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of analogs of the immunomodulary drugs lenalidomide (1) and pomalidomide (2), in which the
amino group is replaced with various isosteres, was prepared and assayed for immunomodulatory activ-
ity and activity against cancer cell lines. The 4-methyl and 4-chloro analogs 4 and 15, respectively, dis-
Received 10 August 2012
Revised 12 October 2012
Accepted 15 October 2012
Available online 27 October 2012
played potent inhibition of tumor necrosis factor-a (TNF-a) in LPS-stimulated hPBMC, potent stimulation
of IL-2 in a human T cell co-stimulation assay, and anti-proliferative activity against the Namalwa lym-
phoma cell line. Both of these analogs displayed oral bioavailability in rat.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Lenalidomide
Pomalidomide
Thalidomide
Tumor necrosis factor-
Immunomodulatory
Anti-cancer
a (TNF-a)
Thalidomide (3), a molecule with diverse pharmacological
activities, was initially marketed as a sedative in the late 1950s
and was also widely used as a treatment for morning sickness in
pregnant women in the United Kingdom, Europe, Canada, and
Australia. Its now well-known tragic teratogenic effects caused
its withdrawal from the world market in the early 1960s. Over
the years, there has been interest in thalidomide for the treatment
of various hematologic malignancies,1 solid tumors,2 and a variety
of inflammatory and autoimmune diseases.3 Recent studies have
uncovered a variety of mechanisms of thalidomide action. It was
reported in 1991 that thalidomide is a selective inhibitor of tumor
immune cell activated as well as the type of stimulus that the cell
receives, provide a rationale for the mechanism of thalidomide
action in the context of autoimmune and inflammatory disease
states. Other pharmacologic activities of thalidomide include its
inhibition of angiogenesis7 and its anti-cancer properties.8 In the
late 1990’s it was reported that thalidomide is efficacious for the
treatment of multiple myeloma (MM), a hematological cancer
caused by growth of tumor cells derived from the plasma cells in
the bone marrow.1 Thalidomide received FDA approval in 2006
for the treatment of newly diagnosed MM. Although the cellular
pharmacology of thalidomide and its analogs had been well stud-
ied, its mechanism of action was not understood until 2010, when
the molecular target of thalidomide responsible for its teratogenic
effects was shown to be a protein named cereblon, that acts as a
co-receptor for an E3 ubiquitin ligase complex including DNA dam-
age binding protein 1 (DDB1) and the E3 ligase cullin 4A (CUL4A).9
Since that initial discovery, cereblon has been reported to also
mediate the anti-MM and T cell costimulatory activities of lenalid-
omide and pomalidomide.10
necrosis factor-
a
(TNF-a
) production in lipopolysaccharide (LPS)
is a key pro-inflammatory
stimulated human monocytes.4 TNF-
a
cytokine, and elevated levels have been linked with the pathology
of a number of inflammatory and autoimmune diseases including
rheumatoid arthritis, Crohn’s disease, aphthous ulcers, cachexia,
graft versus host disease, asthma, ARDS and AIDS.5 Another biolog-
ical activity of thalidomide is its ability to co-stimulate T cells that
have been partially activated by the
T
cell receptor.6 Co-
stimulation results in T cell activation and facilitates an antigen-
specific effector response. Taken together the immunomodulatory
properties of thalidomide, which are dependent on the type of
A medicinal chemistry program to optimize the immunomodu-
latory properties of thalidomide and reduce its side-effects led to
the discovery of lenalidomide (1), which is a potent immunomod-
ulator that is ꢀ800 times more potent as an inhibitor of TNF-
a in
LPS-stimulated hPBMC.11 In the US, lenalidomide was approved
by the FDA in 2005 for low- or intermediate-1-risk myelodysplastic
⇑
Corresponding author. Tel.: +1 908 673 9587; fax: +1 908 673 2792.
E-mail address: aruchelman@celgene.com (A.L. Ruchelman).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.071

A. L. Ruchelman et al. / Bioorg. Med. Chem. Lett. 23 (2013) 360–365
361
O
O
O
O
chemotherapy. Lenalidomide also possesses anti-angiogenic prop-
erties and decreases paracrine secretion of key mediators of angi-
ogenesis.20 In addition to its approved indications of MM and
MDS, clinical studies have also demonstrated that lenalidomide
has efficacious activity in chronic lymphocytic leukemia (CLL)21
and non-Hodgkin’s lymphoma (NHL).22
O
O
NH
NH
NH
N
O
N
O
N
O
O
NH2
NH2
O
1
2
3
, Thalidomide
, Lenalidomide
, Pomalidomide
Structural optimization of thalidomide, 3 also led to the discov-
ery of pomalidomide (2), which was 10-fold more potent than
Figure 1. Structures of lenalidomide (1), pomalidomide (2), and thalidomide (3).
lenalidomide as a TNF-a
inhibitor and IL-2 stimulator.11 Pomalido-
mide is currently undergoing late-stage clinical development for
treatment of multiple myeloma and myeloproliferative neo-
plasm-associated myelofibrosis.23 In clinical trials for multiple
myeloma, pomalidomide has been shown to be effective in over-
coming resistance to lenalidomide and thalidomide, as well as
the proteosome inhibitor bortezomib.24
syndromes (MDS) associated with a chromosome 5q31 deletion,
with or without additional cytogenetic abnormalities, and in
2006 for the treatment of patients with multiple myeloma (MM)
who have received at least one prior therapy. Lenalidomide pos-
sesses direct tumoricidal effects that have been linked to cereblon
binding, resulting in inhibition of expression of tumor oncogenes
such as IRF4 and C-MYC with the attendant induction of tumor sup-
pressor genes,12 cell cycle arrest,13actin polymerization and the
relocalization of membrane proteins leading to cytoskeletal reor-
ganization,14 and inhibition of autocrine cytokines.15 In multiple
myeloma, lenalidomide treatment leads to caspase activation and
resultant tumor cell apoptosis.16 Lenalidomide also has profound
immunomodulatory properties, including restoration of immune
synapse formation,17 augmentation of natural killer cell cytotoxic-
ity,18 and inhibition of regulatory T cells.19 These actions result
in an immune-mediated tumor cell killing that is in direct
contrast to the immune suppression which typifies conventional
Agents 1, 2, and 3 are structurally similar, with all three sharing
the common
a-(isoindolinone-2-yl)-glutarimide core structure
(Fig. 1). A previous report described the activity of positional iso-
mers of 1 and 2.10 It was shown that analogs possessing an amino
group residing at the 4-position of the isoindolinone or isoindolin-
edione (phthalimide) ring displayed potent inhibition of TNF-
a
with the other isomers showing much reduced activity. If the ami-
no group was moved to the 5-position of the phthalimide, or to the
5-, 6-, or 7-positions of the isoindolinone ring, the resulting analogs
had IC50s of P100
success of lenalidomide and pomalidomide,
l
M as TNF-
a
inhibitors. In light of the clinical
more detailed
a
O
O
CO2H
CO2CH3
CO2CH3
CH2Br
a
b
c
NH
R
R
R
N
R
O
29 a-m
30 a-m
4
5
6
7
; R = 4-CH3, ; R = 5-CH3, ; R = 6-CH3, ; R = 7-CH3
15; R = 4-Cl, 16; R = 5-Cl, 17; R = 6-Cl, 18; R = 7-Cl
19; R = 4-OCH3, 20; R = 5-OCH3, 21; R = 7-OCH3
a
b
c
d
; R = 4-CH3, ; R = 5-CH3, ; R = 6-CH3, ; R = 7-CH3
e; R = 4-Cl, f; R = 5-Cl, g; R = 6-Cl, h; R = 7-Cl
i; R = 4-OCH3, j; R = 5-OCH3, k; R = 7-OCH3
22
23
; R = 4-CF3, ; R = 7-CF3
l
m
; R = 4-CF3, ; R = 7-CF3
Scheme 1. Reagents and conditions: (a) CH3I, NaHCO3, DMF or HC(OCH3)3, 79–99%; (b) NBS, AIBN, MeCN or CCl4,15–80%; (c)
a-aminoglutarimide HCl, TEA, DMF, 31–47%.
O
O
O
O
NH
NH
a
N
O
N
O
NH2
OH
1
11
CO2CH3
CH2Br
CO2CH3
CO2CH3
b
c
AcO
O
HO
AcO
31a-b
32a-b
O
CO2CH3
CH2Br
d
e
NH
HO
HO
N
O
33a-b
12
; R = 5-OH
13; R = 6-OH
a
; R = 5-OH
b; R = 6-OH
OH
OH
OH
O
O
e
CO2CH2CH3
CH2Br
c
CO2CH2CH3
NH
N
O
34
14
, 63–69%; (d) NH4OAc, MeOH, H2O or Amberlyst 15, 63–73%; (e) a-
Scheme 2. Reagents and conditions: (a) NaNO2, HCl, H2O, 2.4%; (b) Ac2O, 58–95%; (c) NBS, CCl4, h
t
aminoglutarimide HCl, TEA, DMF or MeCN, 16–47%.

362
A. L. Ruchelman et al. / Bioorg. Med. Chem. Lett. 23 (2013) 360–365
CO2CH3
CO2CH3
CO2CH3
CH2Br
a
c
b
I
TMS
TMS
35
36
O
O
N
O
O
O
O
NH
NH
NH
d
e
N
O
O
N
O
37
38
10
TMS
Scheme 3. Reagents and conditions: (a)TMSA, Pd(PPh3)4, CuI, TEA, 85%; (b) NBS, (PhCO2)2, PhH, 89%; (c)
a
-aminoglutarimide HCl, TEA, DMF, 49%; (d) CsF, DMF, 85%; (e) 10%
Pd-C, H2, DMF, 97%.
CHO
CO2H
a
c
b
39
40
O
NEt2
O
NEt2
CH2Br
O
O
NH
d
e
N
O
41
42
8
Scheme 4. (a) Cl2CHOCH3, TiCl4, CH2Cl2, 90%; (b) KMnO4, Me2CO, H2O; (c) (i) (COCl)2, CH2Cl2, (ii) Et2NH, 71%; (d) (i) s-BuLi, THF, (ii) B(OMe)3, (iii) Br2, 96%; (e) (i)
a-
aminoglutarimide HCl, TEA, MeCN, (ii) AcOH, 72%.
O
O
CO2H
NEt2
NEt2
CHO
a
c
b
43
44
O
O
O
O
NH
d
e
NEt2
NEt2
N
O
OH
OMs
45
46
9
Scheme 5. (a) (i) (COCl)2, CH2Cl2, (ii) Et2NH, 96%; (b) (i) s-BuLi, TMEDA, THF, (ii) DMF, 50%; (c) NaBH4, MeOH, 97%; (d) MsCl, TEA, CH2Cl2, 74%; (e) (i) a-aminoglutarimide HCl,
TEA, MeCN, (ii) AcOH, 52%.
examination of the structure–activity relationships (SAR) of this
remarkable class of compounds is herein reported. The present
study reports the results of isosteric replacement of the amino
moiety of 1 and 2, and presents the synthesis and activity of these
analogs of 1 and 2.
Scheme 1 depicts the standard synthetic pathway used to
access isoindoline analogs of 1. Appropriately substituted
o-methyl benzoic acids were converted to the corresponding
methyl esters and then subjected to NBS-mediated benzylic radical
bromination. The generated benzyl bromide benzoate esters are
aminoglutarimide in the presence of base. The synthesis of
hydroxy-substituted analogs 11–14 is shown in Scheme 2. The
4-hydroxy analog 11 could be obtained in low yield by diazotiza-
tion/hydrolysis of 1. During the synthesis of the 5- and 6-positional
isomers, it was found that the bromination was hindered by the
free phenol but after protection as the respective acetates, the bro-
mination performed adequately and generated the requisite benzyl
bromides in 63–69% yield. Deprotection of the phenol and reaction
with
a-aminoglutarimide delivered the targeted analogs 12–13.
For synthesis of the 7-hydroxy isomer it was found that when
set up for SN2 displacement/cyclization upon treatment with
a-
the ethyl ester was used, phenol protection was unnecessary. To

A. L. Ruchelman et al. / Bioorg. Med. Chem. Lett. 23 (2013) 360–365
363
O
O
lenalidomide (1) and pomalidomide (2): inhibition of TNF-
a
levels
O
NH
in LPS-stimulated human PBMC, potentiation of IL-2 levels in anti-
CD3 stimulated human T cells, and anti-proliferative activity
against Namalwa CSN.70 cells.26 The Namalwa cells are a chromo-
some 5 deleted B cell Burkitt’s lymphoma cell line, selected for
screening as a measure of the compounds’ anti-cancer activity
since it has previously been observed to be the cell line most
responsive to lenalidomide from among a panel of hematopoietic
tumor cell lines with and without chromosome 5 deletions.27 As
noted above, the molecular target(s) of lenalidomide was until re-
cently unknown and there is still no standard quantitative bio-
chemical assay for intrinsic activity. The assays used to measure
bioactivity in this study are cellular assays with TNF inhibition
and IL-2 stimulatory assay using primary human cells that confer
direct human relevance to the assays. As a result, the ability to
reach the target is a prerequisite for activity, and low activity in
the assays may reflect either poor intrinsic activity or inability of
the compound to reach the target.
Biological activity data for isoindolinone analogs of 1 are shown
in Table 1. Compound 1 itself inhibited TNF-a with IC50 of 100 nM
and showed stimulation of T cells with an EC50 of 150 nM. Com-
pared to its activity in these assays of immunomodulation, 1 dis-
played similar potency in the Namalwa anti-proliferation assay,
with IC50 of 360 nM. Isosteric replacement of the amino group in
1 with a methyl group (i.e., 4) provided a 10-fold enhancement
a
O
N
O
R
R
O
O
24-28
Scheme 6. (a)
a-Aminoglutarimide HCl, NaOAc, HOAc, 27–82%.
prepare the 4-ethyl analog 10, methyl 3-iodo-2-methylbenzoate
was converted to the TMS-protected acetylene under Sonogashira
conditions in high yield (Scheme 3). After benzylic bromination
and reaction with
a-aminoglutarimide, the TMS group was re-
moved with CsF and the triple bond was hydrogenated to provided
the targeted ethyl analog. For the preparation of the 4,5-dimethyl
analog 8 (Scheme 4), 1,2,3-trimethylbenzene was formylated using
a,a
-dichloromethyl methyl ether in the presence of TiCl4,25 and the
resulting benzaldehyde was oxidized to the carboxylic acid using
KMnO4. Following conversion to the diethyl amide, directed lateral
lithiation and quench with trimethylborate followed by bromine
treatment delivered the desired benzyl bromide 42 in 96% yield.
After reaction with
a-aminoglutarimide and ring closure, the
product was recrystallized from acetic acid, providing the product
in 72% yield from the benzyl bromide 42. Scheme 5 shows the
synthesis of 4,6-dimethylisoindoline 9. 3,5-Dimethylbenzoic acid
was converted to the diethyl amide 43, and directed ortho
lithiation followed by DMF quench gave the o-formyl amide
in 50% yield. The formyl group was reduced with sodium
borohydride and the resulting benzyl alcohol was mesylated under
of TNF-a and IL-2 activity (IC50 and EC50 were both 10 nM), and a
similar improvement in Namalwa anti-proliferative activity as well
(IC50 13 nM). Thus the heteroatom lone pair and H-bond donating
ability do not appear to be a prerequisite for these biological activ-
ities. Moving the methyl group to the 5-, 6-, or 7-position resulted
standard conditions to yield mesylate 46. Reaction of 46 with
a-
in a significant drop-off in activity measured by the TNF-a and IL-2
aminoglutarimide and recrystallization from acetic acid gave the
targeted analog in 52% combined yield. Finally, the phthalimide
analogs 24–28 were easily accessed in a single step from commer-
cially available appropriately substituted phthalic anhydrides by
assays. This finding is in agreement for the SAR reported previously
for 1;10 namely that substitution was required at the 4-position of
the benzo ring for the most potent activity and that positional iso-
mers showed decreased activity. Compounds 8 and 9 possess an
additional methyl substitution while still having the 4-methyl sub-
stituent on the benzo ring of 4, and these compounds also showed
treatment with
a-aminoglutarimide in a medium of sodium
acetate in acetic acid (Scheme 6).
In this publication we will report on three assays used to test
these novel analogs for biological activities characteristic of
reduced activity with limited if any effects at 10 lM in all three
Table 1
TNF-
a inhibition, IL-2 stimulation, and Namalwa anti-proliferative activity of lenalidomide (1) and isoindolinone analogs 4–23
O
O
NH
N
R
O
1, 4-23
M)a
Compd
Substitution
TNF-a
IC50
(l
IL-2 EC50
(
l
M)a
Namalwa IC50 (l
M)a
1
4
5
6
7
4-NH2
4-CH3
5-CH3
6-CH3
7-CH3
4,5-(CH3)2
4,6-(CH3)2
4-C2H5
4-OH
5-OH
6-OH
7-OH
4-Cl
5-Cl
6-Cl
7-Cl
4-OCH3
5-OCH3
7-OCH3
4-CF3
0.10
0.010
>10
>10
>10
>10
>10
3.5
0.055
>1
>1
>1
0.15
0.010
>10
>10
>10
>10
>10
1.1
0.045
>1
>1
>1
0.021
>1
0.36
0.013
>10
>10
>10
>10
>10
>10
0.18
>1
>1
>1
0.067
>1
>1
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
0.079
>1
>1
>1
>1
>1
0.65
>1
>1
>1
>1
>1
>1
>1
>1
1.2
>1
>1
>1
>1
>1
>1
7-CF3
a
IC50 and EC50values are the means of at least three independent determinations.

364
A. L. Ruchelman et al. / Bioorg. Med. Chem. Lett. 23 (2013) 360–365
Table 2
TNF- inhibition, IL-2 stimulation, and Namalwa anti-proliferative activity of pomalidomide (2), thalidomide (3) and phthalimide analogs 24–28
a
O
O
NH
N
R
O
O
2,3, 24-28
Compd
Substitution
TNF-a
IC50
(l
M)a
IL-2 EC50
(
l
M)a
Namalwa IC50 (l
M)a
2
3
4-NH2
—
4-OH
4-CH3
4-Cl
0.013
77
>1
0.10
>1
>1
0.0080
>10
>1
0.057
>1
>1
0.030
nd
>1
nd
>1
24
25
26
27
28
4-OCH3
4-C2H5
>1
>10
2.9
>10
nd = not determined.
a
IC50 and EC50values are the means of at least three independent determinations.
Table 3
PK parameters in fasted male CD-IGS rats after oral administration in 0.5% CMC/0.25% Tween 80 suspension formulation in a volume of 5 mL/kg
Compd
Structure
Dose (mg/kg)
Cmax (ng/mL)
AUC (ng-h/mL)
Tmax (h)
O
O
NH
4
N
10
4600
29,000
2.0
O
O
O
NH
N
O
15
25
10
10
2800
440
24,000
2200
1.7
2.5
Cl
O
O
NH
N
O
O
assays. This result demonstrates that not only is there a require-
ment for the 4-substitution in this series, but there is an additional
requirement for no methyl group at the 5- or 6-positions for opti-
mal activity in these analogs. In compound 10, the methyl group of
4 was homologated to an ethyl group. Surprisingly, this relatively
minor modification resulted in >100-fold decline in potency. Evi-
dently there is a strict threshold for some property of the group
at site, violation of which results in a dramatic decline in observed
potency.
that it is not important whether the atom at the 4-position of the
benzo ring is carbon or a heteroatom. The results for the trifluoro-
methyl analog 22 showed reduced activity compared to methyl
compound 4. Although 22 is a closely related structural analog of
lenalidomide, it showed significantly less activity. The reason for
its reduced potency is not currently understood.
Table 2 presents biological activity data for pomalidomide (2),
thalidomide (3), and for phthalimide analogs 24–28. In a previous
report,10 we reported that pomalidomide is approximately 10-fold
In addition to the methyl group, the use of other amine bioisos-
teres was also investigated. The 4-hydroxy analog 11 and the
4-chloro analog 15 both showed activity that was intermediate be-
tween 1 and 4. Like 1 and 4, these analogs also possessed improved
more potent than 2 as a TNF-a inhibitor. As shown in Table 2, it
also has 10-fold greater potency in the IL-2 and Namalwa assays,
with IC50 or EC50 values of ꢀ10 nM in all three assays. However,
the new phthalimide analogs 24–26 were less active than the cor-
responding isoindolinone analogs 4, 11, and 15. Of the three, only
Namalwa IC50s that were similar to their TNF IC50 and IL-2 EC50
,
suggesting that they are relatively non-selective among these
activities or that the activities are linked. As was the case with
the methyl series, positional isomers in which the hydroxy or
chloro group was moved around the benzo ring displayed less
the methyl analog was active at sub lM concentrations. The rever-
sal of relative activity of the isoindolinone and phthalimide analogs
is puzzling. It is possible that differences in cellular permeability or
another physicochemical property takes on increasing importance
in the case of the phthalimides. However, whether or not the re-
duced activity of 24–26 reflects the compounds’ intrinsic activity,
it is clear that these compounds are much less potent. Determina-
tion of the intrinsic activity awaits the availability of a quantitative
biochemical assay.
activity, with IC50 or EC50 >10 lM in all three assays. Methoxy ana-
log 19 was roughly ten-fold less active than the hydroxy com-
pound 4; its activity was similar to that observed for the ethyl
analog 10, which is in accord with the SAR discussed above in
two ways. First, adding an additional carbon to the atom bound
to the 4-position of the phenyl ring was detrimental; and second,
the similar potency of the ethyl and methoxy analogs illustrates
Compounds 4, 15, and 25 were selected for pharmacokinetic
evaluation in rat (Table 3). All compounds were administered

A. L. Ruchelman et al. / Bioorg. Med. Chem. Lett. 23 (2013) 360–365
365
orally at a dose of 10 mg/kg.28 The two isoindolinones 4 and 15 dis-
played good oral exposure while the phthalimide 25 had only
moderate exposure. The methyl isoindolinone 4 achieved a Cmax
of 4600 ng/mL at 2 h, and had an AUC of 29,000 ng-h/mL. Chloro
isoindolinone 15 had a similar AUC (24,000 ng-h/mL), although
Cmax was somewhat lower (2800 ng/mL). On the other hand, the
methyl phthalimide 25 showed a ꢀ10-fold lower Cmax and AUC.
In summary, the isosteric replacement the amino moiety of
lenalidomide and pomalidomide resulted in the identification of
several potent analogs with drug-like physicochemical properties.
The concept of isosterism was used to guide compound design. The
amino group of lenalidomide could be replaced with methyl or
chloro moieties to provide new analogs with potent activity and
good pharmacokinetic performance in a rodent model. Lenalido-
mide has demonstrated a variety of pharmacological activities
and has been used successfully to treat thousands of patients;
the identification and characterization of other analogs may lead
to other therapeutics. The clinical activity of thalidomide in oncol-
ogy and inflammatory diseases suggest that pre-clinical study of
the new analogs may uncover differences in the profile of biologi-
cal activities, differentiate new compounds, and point to areas of
clinical activity for compounds in these series.8 Future publications
will expand on the SAR reported here.
Hideshima, T.; Lentzsch, S.; Young, G.; Tai, Y.-T.; Lin, B.; Podar, K.; Gupta, D.;
Chauhan, D.; Treon, S.; Richardson, P.; Schlossman, R.; Morgan, G.; Muller, G.;
Stirling, D.; Anderson, K. Blood 2001, 98, 210; (c) Wu, L.; Adams, M.; Carter, T.;
Chen, R.; Muller, G.; Stirling, D.; Schafer, P.; Bartlett, J. Clin. Cancer Res. 2008, 14,
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19. Galustian, C.; Meyer, B.; Labarthe, M.-C.; Dredge, K.; Klaschka, D.; Henry, J.;
Todryk, S.; Chen, R.; Muller, G.; Stirling, D.; Schafer, P.; Bartlett, J.; Dalgleish, A.
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References and notes
in 96-well flat-bottom Costar tissue culture plates in triplicate. Cells were
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compounds were dissolved in DMSO and further dilutions were done in
culture medium immediately before use. The final DMSO concentration in
all assays was 0.25%. Compounds were added to cells 1 h before LPS
stimulation. Cells were incubated for 18–20 h at 37 °C in 5% CO2 and
supernatants were then collected, diluted with culture medium and assayed
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flat-bottom plates were coated with anti-CD3 antibody OKT3 at 5
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with complete medium 100 L/well just before T cells were added. Cultures
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were incubated at 37 °C 5% CO2 for 2–3 days, and supernatants analyzed for
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produced in the presence of 10
calculated using non-linear regression,
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CC-4047, and EC50 values were
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constraining the top to 100% and bottom to 0%, allowing variable slope.
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6000 cells/75
samples were done in triplicate. Cells were incubated at 37 °C in
humidified incubator at 5% CO2 for 72 h. One microcurie of 3H-thymidine
was added to each well and cells were incubated again at 37 °C in
humidified incubator at 5% CO2 for an additional 6 h. The cells were
harvested onto UniFilter GF/C filter plates (Perkin Elmer) using cell
harvester (Tomtec) and the plates were allowed to dry overnight. Microscint
20 (Packard) (25 l/well) was added and plates were analyzed in TopCount
lL medium (RPMI-1640 10% FCS 1% Pen/Strep) per well. All
a
a
a
l
NXT (Packard). Each well was counted for one minute. The percent
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28. CD-IGS rats (male, jugular vein-cannulated (JVC), weight range: 200–225 g)
supplied by Charles River Laboratories were used in this study. Test articles
were administered as a single po administration at 10 mg/kg in a 0.5% CMC/
0.25% Tween 80 suspension formulation, in
a volume of 5 mL/kg. Serial
blood samples were obtained via the cannulae at specified time points:
30 min, 1, 2, 4, 9, and 24 h post-dose, deposited into heparinized vacutainer
tubes and stored on ice after collection. The harvested whole blood was then
centrifuged at ꢀ9000 rpm for ꢀ10 min within 0.5 h to separate plasma. An
aliquot of plasma was then transferred to a 1.5 mL Eppendorf vial, an equal
volume of Sorensen’s buffer (250 mM citrate buffer, pH 1.5) was added,
samples were vortexed and then placed on dry ice. Following terminal
sample collection, all samples were transferred to
processing. The pre-diluted plasma samples (50
a
À20 °C freezer until
16. Mitsiades, N.; Mitsiades, C.; Poulaki, V.; Chauhan, D.; Richardson, P.;
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Jagannath, S.; Dhodapkar, M. Blood 2006, 108, 618; (b) Davies, F.; Raje, N.;
l
L) were processed by
addition of 2.5 volumes of methanol containing internal standard
(proprietary compound), filtration of the mixture through a Captiva™ 96-
well filtration plate (0.45 lm; Varian, Inc., Palo Alto, CA), and an aliquot of
the filtrate was injected into
spectrometer (LC/MS/MS).
a liquid chromatography/tandem mass